Targeting agent

Abstract

This provides a means for targeting substances to motor neurons. [Solution] The present invention provides a targeting agent for motor neuron synapses, comprising an antibody capable of binding to the intravesicular domain of synaptotagmin 2.

Description

Technical Field

[0001] The present invention relates to a targeting agent for motor neurons, a motor neuron visualization agent, a composition containing the same, a targeting method using them, a motor neuron visualization method, a method for preventing or treating a condition or disease, and the like.

Background Art

[0002] Nerves are composed of the central nervous system and the peripheral nervous system, and regulate various emotions, functions of muscles and internal organs, and the like. Such nerve functions may decline due to nerve damage, diseases, aging, etc. In such cases, physical and mental health may be impaired, and it may have a great impact on leading a social life. Therefore, maintaining and improving nerve function can be said to be an extremely important issue because it directly contributes to maintaining and improving the quality of life (QOL).

[0003] The central nervous system and the peripheral nervous system are each composed of nerve cells, and these cells exchange signals with each other via synapses. A synapse is a junction including a gap formed between the axon terminal (presynaptic part) of a nerve cell and the dendrite of another nerve cell or a cell such as a skeletal muscle or an organ (postsynaptic part), and a chemical substance released from the presynaptic part binds to a receptor present in the postsynaptic part to transmit a signal. Synapse formation is triggered by the interaction of specific membrane proteins expressed in the presynaptic and postsynaptic parts.

[0004] In recent years, compounds and peptides have been developed that can maintain and improve nerve function by promoting synapse formation. Patent Document 1 describes that a specific peptide has dendritic elongation promoting and synapse formation promoting effects in primary cortical neuron (PCN) culture cells, and that such peptides can be used to treat mild cognitive impairment or early dementia. Patent Document 2 describes that C-terminal fragment β (CTFβ), produced when amyloid precursor protein (APP) is cleaved by β-secretase, promotes synapse formation, and that CTFβ can be used to treat neurodegenerative diseases, etc.

[0005] Furthermore, Patent Document 3 describes a method for culturing motor neurons having a presynaptic terminal using microbeads on which LRRTM molecules or fusion proteins containing these molecules are immobilized on their surface. Thus, with the development of technologies that allow for simple in vitro screening of the effects of drugs on motor neuron function, it is expected that a large number of compounds and drug candidates capable of acting on motor neurons will be available in the future. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] Japanese Patent Publication No. 2012-092048 [Patent Document 2] Japanese Patent Publication No. 2008-143867 [Patent Document 3] WO2021 / 006075 [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] For the compounds and drugs described above to act on motor neurons, controlled delivery of the drugs to the target motor neurons is necessary. Furthermore, it is important to be able to determine the location of the targets on which the drugs can act. However, until now, the delivery of substances to nerve cells such as motor neurons and the visualization of delivery sites have not been investigated.

[0008] Therefore, the object of the present invention is to provide a means for targeting a substance to motor neurons and a means for visualizing the targeting site. [Means for solving the problem]

[0009] The inventors focused on the fact that specific proteins are expressed on the membrane of synaptic vesicles and, after diligent research to solve the above problem, discovered that it is possible to target desired substances to motor neurons by using an antibody that binds to the intravesicular domain (N-terminal portion) of synaptotagmin 2. Furthermore, they found that similar effects can be obtained when using antibodies that bind to other synaptic vesicle membrane proteins, thus completing the present invention.

[0010] In other words, the present invention encompasses the following:

[0011] [1] A targeting agent for motor neurons, comprising an antibody capable of binding to the intravesicular domain of membrane proteins present in synaptic vesicles of motor neurons. [2] The targeting agent according to [1], further comprising a labeled substance and / or a physiologically active substance. [3] The targeting agent according to [2], comprising a conjugate of the antibody and the labeled substance and / or the physiologically active substance. [4] The targeting agent according to any one of [1] to [3], wherein the membrane protein is a human-derived protein. [5] The targeting agent according to any one of [1] to [4], wherein the membrane protein comprises one protein selected from the group consisting of synaptotagmin 2, synaptic vesicle glycoprotein 2A, synaptogyrin 1, synaptophysin, and synaptotagmin 1. [6] The targeting agent according to any one of [1] to [5], wherein the intravesicular domain is a domain containing four or more amino acids. [7] The targeting agent according to [5] or [6], wherein the intravesicular domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7-12, 15, 16, 19, 20, and 23; an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7-12, 15, 16, 19, 20, and 23; or an amino acid sequence having 90% or more sequence identity with the amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7-12, 15, 16, 19, 20, and 23. [8] The targeting agent according to any one of [2] to [7], wherein the labeled substance is a fluorescent molecule. [9] A targeting agent according to any one of [2] to [8], which is a motor neuron visualization agent and comprises the labeling substance.

[10] The targeting agent according to any one of [2] to [9], wherein the physiologically active substance is one or more selected from the group consisting of synapse formation promoters, synapse maintenance agents, muscle strengthening agents and nerve cell function modifiers.

[11] A targeting agent described in any of [1] to

[10] that is taken up into cells by endocytosis. A pharmaceutical composition comprising a targeting agent as described in any of

[12] [2] to

[11] . A method for targeting motor neurons with a labeled substance and / or a bioactive substance, comprising the steps of: contacting a motor neuron with a targeting agent and / or a pharmaceutical composition described in any of

[13] [2] to

[11] ; and delivering the targeting agent and / or the pharmaceutical composition to the synapse of the motor neuron. A method for visualizing motor neurons, comprising the steps of bringing a motor neuron visualization agent described in

[14] [9] into contact with motor neurons, delivering the motor neuron visualization agent to the synapse of the motor neurons, and detecting the signal of the labeling substance. A method for preventing or treating a condition or disease, comprising the steps of bringing a targeting agent described in any of

[15] [2] to

[11] into contact with a motor neuron, and delivering the targeting agent to the synapse of the motor neuron.

[16] The prevention or treatment method described in

[15] , wherein the condition or disease is a condition or disease exhibiting a decline in nerve function.

[17] The preventive or therapeutic method according to

[15] or

[18] , wherein the condition or disease is a neurological disease or a neuromuscular disease.

[18] The method of prevention or treatment according to any one of

[15] to

[17] , wherein the targeting agent comprises a conjugate of the antibody and the physiologically active substance.

[19] Use of an antibody capable of binding to the intravesicular domain of a membrane protein present in synaptic vesicles of motor neurons in the manufacture of a pharmaceutical product comprising the antibody and a bioactive substance.

[20] 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.

[21] Antibodies capable of binding to the intravesicular domains of membrane proteins present in synaptic vesicles of motor neurons, for use in methods of preventing or treating a condition or disease.

[22] An antibody capable of binding to the intravesicular domain of a membrane protein present in synaptic vesicles of a motor neuron, for use in a method for targeting labeling substances and / or bioactive substances to motor neurons. This specification includes the disclosures of Japanese Patent Application No. 2022-071453, which forms the basis of the priority claim of this application. [Effects of the Invention]

[0012] According to the targeting agent and targeting method of the present invention, a desired substance can be targeted to motor neurons using an antibody. By using an antibody, the desired substance can be delivered to motor neurons (e.g., synaptic vesicles within the cell). If the substance is a physiologically active substance or a therapeutic agent, symptoms and / or diseases caused by abnormalities in motor neurons can be treated. Furthermore, if the substance is a labeled substance, motor neurons can be visualized. [Brief explanation of the drawing]

[0013] [Figure 1] Figure 1 schematically shows the experimental procedures for the induction of presynaptic terminals and the delivery of antibodies using LRRTM2 beads. When neurospheres are seeded on a plate, neurites extend from the neurospheres. Subsequently, by seeding LRRTM2 beads there, presynaptic terminals are induced on the surface of the LRRTM2 beads from the extended neurites. To such a plate on which presynaptic terminals are induced, a normal rabbit IgG antibody (control) or an anti-synaptotagmin 2 intracellular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) was introduced, and it was examined whether the anti-SYT2 N-terminal antibody was targeted to the presynaptic terminals by stimulation with a chemical substance or by activating nerve cells spontaneously. [Figure 2-1] Figure 2-1 shows fluorescence images in the case of conducting an antibody delivery experiment by spontaneous activity at an antibody concentration of 1 μg / mL. A and B are fluorescence images showing the localization of the administered antibody when using a normal rabbit IgG antibody (A) and an anti-synaptotagmin 2 intracellular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) (B) as the antibody, respectively. In the figure, the scale bar is common to A and B and indicates 100 μm. [Figure 2-2] Figure 2-2 shows fluorescence images in the case of conducting a delivery experiment of an anti-synaptotagmin 2 intracellular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) by spontaneous activity at an antibody concentration of 1 μg / mL. A and B are fluorescence images showing the localization of the anti-SYT2 N-terminal antibody in LRRTM2 beads with sparse neurites (A) and LRRTM2 beads with dense neurites (B), respectively. In the figure, the dashed circle indicates the position of the LRRTM2 beads. TUJ1 refers to a stained image showing the signal of βIII tubulin, and the scale bar is common to A and B and indicates 10 μm. [Figure 3]Figure 3 is a diagram showing fluorescence images when an antibody delivery experiment by spontaneous activity was conducted at an antibody concentration of 10 μg / mL. A and B are fluorescence images showing the localization of the administered antibody when normal rabbit IgG antibody (A) and anti-synaptotagmin 2 intracellular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) (B) were used as the antibody, respectively. In the figure, the scale bar is common to A and B and indicates 100 μm. [Figure 4] Figure 4 is a diagram showing fluorescence images when an antibody delivery experiment by stimulating neurons with 4-aminopyridine for 10 minutes was conducted. A and B are fluorescence images showing the localization of the administered antibody when normal rabbit IgG antibody (A) and anti-synaptotagmin 2 intracellular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) (B) were used as the antibody, respectively. In the figure, the scale bar is common to A and B and indicates 100 μm. [Figure 5] Figure 5 is a diagram showing fluorescence images when an antibody delivery experiment by stimulating neurons with 4-aminopyridine for 30 minutes was conducted. A and B are fluorescence images showing the localization of the administered antibody when normal rabbit IgG antibody (A) and anti-synaptotagmin 2 intracellular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) (B) were used as the antibody, respectively. In the figure, the scale bar is common to A and B and indicates 100 μm. [Figure 6] Figure 6 is a diagram showing fluorescence images at the neuromuscular junction 12 hours after antibody administration by tail vein injection. A and B are fluorescence images showing the localization of the administered antibody when normal rabbit IgG antibody (A) and anti-synaptotagmin 2 intracellular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) (B) were used as the antibody, respectively. In the figure, α-BgtX refers to a stained image showing the signal of α-bungarotoxin, and SYN1 refers to a stained image showing the signal of synapsin 1. The arrowhead indicates the position 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 μm. [Figure 7]Figure 7 shows fluorescence images of the neuromuscular junction 72 hours after antibody administration by tail vein injection. A and B are fluorescence images showing 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 the antibody, respectively. In the figure, α-BgtX refers to a stained image showing the signal of α-bungarotoxin, and SYN1 refers to a stained image showing the signal of synapsin 1. The arrowhead indicates the location of the neuromuscular junction to which the anti-SYT2 N-terminal antibody was delivered, and the scale bar is common to all images and represents 50 μm. [Figure 8] Figure 8 shows fluorescence images of the neuromuscular junction 12 hours after antibody administration by intraperitoneal injection. A and B are fluorescence images showing 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 the antibody, respectively. In the figure, α-BgtX refers to a stained image showing the signal of α-bungarotoxin, and SYN1 refers to a stained image showing the signal of synapsin 1. The arrowhead indicates the location of the neuromuscular junction to which the anti-SYT2 N-terminal antibody was delivered, and the scale bar is common to all images and represents 50 μm. [Figure 9] Figure 9 shows fluorescence images of the neuromuscular junction 72 hours after antibody administration by intraperitoneal injection. A and B are fluorescence images showing 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 the antibody, respectively. In the figure, α-BgtX refers to a stained image showing the signal of α-bungarotoxin, and SYN1 refers to a stained image showing the signal of synapsin 1. The arrowhead indicates the location of the neuromuscular junction to which the anti-SYT2 N-terminal antibody was delivered, and the scale bar is common to all images and represents 50 μm. [Figure 10]Figure 10 shows electron microscope images of the neuromuscular junction 72 hours after antibody administration by tail vein injection. A and B are electron microscope images showing the localization of administered antibodies, when normal rabbit IgG antibody (A) and anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) (B) were used as the antibodies, respectively. In the figure, the area enclosed by the white line represents the nerve axon terminal of motor neurons, and the area enclosed by the black dashed line represents skeletal muscle cells. The black dots indicate the location of the antibody, and the arrowheads indicate the location of mitochondria. The scale bar is common to both A and B and represents 2 μm. [Figure 11] Figure 11 shows electron microscope images 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 as Figure 10B, and B is an electron microscope image of the region enclosed by the solid black line in A, magnified. In the figures, the black dots indicate the location of the antibody. In Figure A, the region enclosed by the white line shows the nerve axon terminal of a motor neuron, and the region enclosed by the dashed black line shows gastrocnemius cells, which are skeletal muscle cells. The scale bar represents 2 μm. In Figure B, a-c represent synaptic vesicles without antibody (a), synaptic vesicles containing antibody (b), and mitochondria (c), respectively. In Figure B, the scale bar represents 500 nm. [Figure 12] Figure 12 shows fluorescence images 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. In the figure, ChAT refers to the stained image showing the signal of choline acetyltransferase. The dashed line indicates the boundary between the spinal cord anterior horn and white matter, with the spinal cord anterior horn located to the left of the dashed line. The arrowhead illustrates the location of the cell body of the motor neuron to which the anti-SYT2 N-terminal antibody was delivered, and the scale bar is common to all images and represents 100 μm. [Figure 13] Figure 13 shows a magnified fluorescence image of the anterior horn of the spinal cord 72 hours after administration of normal rabbit IgG antibody via tail vein injection. In the figure, ChAT refers to a stained image showing the signal of choline acetyltransferase. The scale bar is common to all images and represents 100 μm. [Figure 14] Figure 14 shows a magnified fluorescence image of the spinal cord anterior horn 72 hours after administration of anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) via tail vein injection. In the figure, ChAT refers to a stained image showing the signal of choline acetyltransferase. The arrowheads illustrate the location of the cell bodies of motor neurons to which the anti-SYT2 N-terminal antibody was delivered, and the scale bar is common to all images and represents 100 μm. [Figure 15] Figure 15 shows fluorescence images of the spinal cord anterior horn 6 to 72 hours after administration of anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) via tail vein injection. The fluorescence signals shown in the figure are signals based on the anti-SYT2 N-terminal antibody. The scale bar in the figure is common to all images and represents 100 μm. [Figure 16] Figure 16 shows fluorescence images of the spinal cord anterior horn 120 to 240 hours after administration of anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) via tail vein injection. The fluorescence signals shown in the figure are signals based on the anti-SYT2 N-terminal antibody. In the figure, the scale bar is common to all images and represents 100 μm. [Figure 17] Figure 17 shows fluorescence images of a control antibody group into which a conjugate of normal rabbit antibody and monomethyl auristatin E (MMAE) was introduced. The fluorescence signals shown in the figure are based on βIII tubulin (Tuj1) signals. In Figure A, the black dashed circle indicates the location of the neurosphere. Figures B and C show magnified images of the area within the white frame in Figure A. In Figure A, the scale bar represents 1 mm, and in Figures B and C, the scale bar represents 100 μm. [Figure 18]Figure 18 shows fluorescence images of a group of SYT2 antibodies 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 fluorescence signals shown in the figure are based on βIII tubulin (Tuj1) signals. Figures B and C show magnified images of the area within the white frame in Figure A. In Figure A, the scale bar represents 1 mm, and in Figures B and C, the scale bar represents 100 μm. [Figure 19] Figure 19 shows a graph quantifying the pharmacological effects based on monomethyl auristatin E (MMAE). In the figure, the relative axonal volume is a value standardized with the result using a conjugate of control normal rabbit antibody and MMAE (in the figure, "Cont. IgG-MMAE") set to 100%. In the figure, the dashed line indicates the position of 100% relative axonal volume, "MMAE" shows the results for the monotherapy group where MMAE was introduced alone, and "α-SYT2 IgG-MMAE" shows the results for the SYT2 antibody group where a conjugate of anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) and monomethyl auristatin E (MMAE) was introduced. In the figure, error bars indicate the standard error, "*" indicates p<0.05, and "***" indicates p<0.001. [Figure 20] Figure 20 shows fluorescence images of normally firing neurons cultured under the same conditions as in Figure 18, after introducing a conjugate of anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) and monomethyl auristatin E (MMAE). The fluorescence signals shown in the figure are based on βIII tubulin (Tuj1). The scale bar in the figure represents 1 mm. [Figure 21] Figure 21 shows fluorescence images of synaptic dysynaptic cells cultured under conditions that did not induce presynaptic formation, by introducing a conjugate of anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) and monomethyl auristatin E (MMAE). The fluorescence signals shown in the figure are based on βIII tubulin (Tuj1) signals. The scale bar in the figure represents 1 mm. [Figure 22] Figure 22 shows fluorescence images of neurons cultured under low-temperature conditions that inhibit endocytosis, after introducing a conjugate of anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) and monomethyl auristatin E (MMAE). The fluorescence signals shown in the figure are based on βIII tubulin (Tuj1). The scale bar in the figure represents 1 mm. [Figure 23] Figure 23 is a graph showing the quantitative pharmacological effects based on monomethyl auristatin E (MMAE) delivered by an anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody). In the figure, relative axonal volume is a value standardized with the synapse-non-synaptic group, cultured under conditions where presynaptic formation is not induced, set to 100%. In the figure, the dashed line indicates the position of 100% relative axonal volume, "α-SYT2 IgG-MMAE" indicates the presence or absence of introduction of the anti-SYT2 N-terminal antibody and MMAE conjugate, "synapse" indicates the presence or absence of synapse formation induction, and "endocytosis" indicates the presence or absence of endocytosis. In the figure, error bars indicate the standard error, and "**" indicates p<0.01. [Figure 24]Figure 24 shows graphs quantifying the pharmacological effects based on monomethyl auristatin E (MMAE) delivered by various antibodies. The results are standardized with the results using a conjugate of a control normal rabbit antibody and MMAE (labeled "Cont." in the figure) set to 100%. In the figure, the dashed line indicates the position of 100% relative axonal volume. "α-SYT2" represents the conjugate of anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) and MMAE, "α-SYP" represents the conjugate of anti-synaptophysin intravesicular domain antibody and MMAE, "α-SYNGR1" represents the conjugate of anti-synaptogyrin 1 intravesicular domain antibody and MMAE, "α-SYT1" represents the conjugate of anti-synaptotagmin 1 intravesicular domain antibody and MMAE, and "α-SV2A" represents the conjugate of anti-synaptic vesicular protein 2 intravesicular domain antibody and MMAE. In the figure, the error bars indicate the standard error; "*" indicates p<0.05, "**" indicates p<0.01, "***" indicates p<0.001, and "****" indicates p<0.0001. [Modes for carrying out the invention]

[0014] The present invention will be described in detail below.

[0015] <Targeting agent> The present invention relates to a targeting agent for motor neurons (referred to as "the targeting agent of the present invention") which includes an antibody capable of binding to the intravesicular domain of a membrane protein present in synaptic vesicles of motor neurons (referred to as "intravesicular domain antibody").

[0016] A "membrane protein" refers to a protein present on a biological membrane. In this invention, a "membrane protein" specifically refers to a protein present on the membrane of a synaptic vesicle. In this invention, membrane proteins include transmembrane proteins, superficial membrane proteins, and lipid-modified proteins. Superficial membrane proteins and lipid-modified proteins are both proteins that do not have a transmembrane domain.

[0017] The type of membrane protein in the targeting agent of the present invention is not particularly limited as long as it is a protein containing an intravesicular domain, but it is preferably a transmembrane protein.

[0018] In this specification, the term "intravesical domain" of a protein refers to the protein region in a membrane protein that is exposed to the lumen of a synaptic vesicle. The length of the intravesical domain of the membrane protein in the targeting agent of the present invention is not particularly limited. For example, membrane proteins having consecutive intravesical domains of 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 can be used. For example, transmembrane proteins having consecutive intravesical domains of 4 or more amino acids can be preferably used.

[0019] In this specification, "lumen of synaptic vesicle" refers to the space located on the opposite side of the cytoplasm within a synaptic vesicle.

[0020] The lumen of a synaptic vesicle communicates with the extracellular space when the synaptic vesicle fuses with the cell membrane. Therefore, the intravesicular domain in the membrane protein of the targeting agent of the present invention is exposed to the extracellular space when the synaptic vesicle fuses with the cell membrane.

[0021] First, let's explain using synaptotagmin 2 as the membrane protein as an example.

[0022] The present invention relates to a targeting agent for motor neurons, comprising an antibody capable of binding to the intravesicular domain (N-terminal portion) of synaptotagmin 2 (referred to as "anti-SYT2 N-terminal antibody").

[0023] In this specification, "synaptotagmin 2" refers to one of the membrane proteins belonging to the synaptotagmin family. The synaptotagmin family includes 17 proteins in mammals, of which synaptotagmin 2 is mainly expressed on the synaptic vesicle membrane of the presynaptic portion of the neuromuscular junction in peripheral nerves, and is a protein that promotes the fusion of synaptic vesicles and the cell membrane in a calcium ion-dependent manner (see, for example, 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-pass transmembrane protein and contains an intravesicular domain, a transmembrane domain, and a cytoplasmic domain in that order from the N-terminus. The C-terminal cytoplasmic domain has a tandem C2 domain that can bind calcium ions, and this cytoplasmic domain is known to be primarily responsible for membrane fusion.

[0024] The intravesicular domain of synaptotagmin 2 is exposed to the lumen of the synaptic vesicle. As the intracellular calcium ion concentration increases, the synaptic vesicle fuses with the cell membrane, connecting the lumen of the synaptic vesicle to the extracellular space. As a result, the intravesicular domain of synaptotagmin 2 is temporarily exposed to the extracellular space. Subsequently, the cell membrane portion containing synaptotagmin 2 is reabsorbed into the cell by endocytosis as the synaptic vesicle membrane and reused as a synaptic vesicle. At this time, the intravesicular domain of synaptotagmin 2 is once again exposed to the lumen of the synaptic vesicle.

[0025] Specifically, the exemplary human synaptotagmin 2 is a protein consisting of 419 amino acids, represented by the amino acid sequence SEQ ID NO: 1. The locations of each domain are, for example, in SEQ ID NO: 1, the intravesicular domain is the region indicated by the amino acid sequence from positions 1 to 62, the transmembrane domain is the region indicated by the amino acid sequence from positions 63 to 83, and the cytoplasmic domain is the region indicated by the amino acid sequence from positions 84 to 419.

[0026] An example of mouse synaptotagmin 2 is a protein consisting of 422 amino acids, represented by the amino acid sequence SEQ ID NO: 2. In SEQ ID NO: 2, the intravesicular domain is the region indicated by the amino acid sequence from positions 1 to 60, the transmembrane domain is the region indicated by the amino acid sequence from positions 61 to 87, and the cytoplasmic domain is the region indicated by the amino acid sequence from positions 88 to 422.

[0027] Sequence information for synaptotagmin 2 in other organisms can be easily obtained from publicly known databases such as the NCBI database.

[0028] In this specification, "intravesical domain of synaptotagmin 2" refers to all or part of the intravesical domain located in the N-terminal portion of synaptotagmin 2. The entire intravesical domain includes, for example, the region indicated by the amino acid sequence from positions 1 to 62 in SEQ ID NO: 1 (SEQ ID NO: 3) and the region indicated by the amino acid sequence from positions 1 to 60 in SEQ ID NO: 2 (SEQ ID NO: 4). Part of the intravesical domain includes, for example, the region indicated by the partial sequence of any length in SEQ ID NOs: 3 and 4. More specifically, it includes, for example, the N-terminal portion of synaptotagmin 2 having a mutant amino acid sequence in which one or more amino acids are substituted, inserted, deleted, and / or added in the amino acid sequence of SEQ ID NO: 3 to 11 (both identical to SEQ ID NO: 5) and the amino acid sequence of SEQ ID NO: 5. Here, "multiple" refers to two or more, for example, 2 to 6, 2 to 5, 2 to 4, or 2 to 3, preferably 2.

[0029] The targeting agent of the present invention can be an antibody capable of binding to any membrane protein other than synaptotagmin 2 as exemplified above.

[0030] Specific membrane proteins in the targeting agent of the present invention include, in addition to synaptotagmin 2, proteins belonging to any one of the families selected from the group consisting of the synaptotagmin family, major facilitator superfamily, synaptic vesicle glycoprotein 2 family, synaptogyrin family, synaptophysin / synaptobrevin family, synaptobrevin family, vesicle amine transporter family, solute carrier family 5, vesicle glutamate transporter family, amino acid / polyamine transporter family, secreted carrier-related membrane protein family, glutamate transporter subfamily, autophagy-related protein 9 family, glycotransporter family, and vacuolar ATPase subunit S1 family.

[0031] More specifically, in addition to synaptotagmin 2, the membrane proteins in the targeting agent of the present invention include, for example, synaptic vesicle glycoprotein 2A, synaptogyrin 1, synaptophysin, synaptotagmin 1, synaptogyrin 3, vesicle acetylcholine transporter, high affinity choline transporter, vesicle glutamate transporter 1, vesicle glutamate transporter 3, vesicle 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-associated protein 9A, glucose transporter 4, and ATPase H+ transport accessory protein 1. Preferably, the membrane protein in the targeting agent of the present invention comprises any one protein selected from the group consisting of synaptotagmin 2, synaptic vesicle glycoprotein 2A, synaptogyrin 1, synaptophysin, and synaptotagmin 1. In this case, the present invention provides a targeting agent comprising an antibody capable of binding to the intravesicle domain of any one protein selected from the group consisting of synaptotagmin 2, synaptic vesicle glycoprotein 2A, synaptogyrin 1, synaptophysin, and synaptotagmin 1.

[0032] Synaptic vesicle glycoprotein 2A (SV2A) 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 regulation of regulated secretion in nerve cells 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. An exemplary synaptic vesicle glycoprotein 2A is a human-derived protein consisting of 742 amino acids, represented by the amino acid sequence SEQ ID NO: 6.

[0033] In this specification, "intravesical domain of synaptic vesicle glycoprotein 2A" refers to all or part of the intravesical domain of synaptic vesicle glycoprotein 2A. The entire intravesical domain includes, for example, the region indicated by the amino acid sequence from positions 191 to 205 in SEQ ID NO: 6 (sequence: PSAEKDMCLSDSNKG; SEQ ID NO: 7), the region indicated by the amino acid sequence from positions 255 to 262 (sequence: YGTFLFCR; SEQ ID NO: 8), the region indicated by the amino acid sequence from positions 316 to 334 (sequence: PHYGWSFQMGSAYQFHSWR; SEQ ID NO: 9), and the amino acid sequence from positions 469 to 598 (sequence: PDMIRHLQAVDYASRT). This includes the region indicated by KVFPGERVEHVTFNFTLENQIHRGGQYFNDKFIGLRLKSVSFEDSLFEECYFEDVTSSNTFFRNCTFINTVFYNTDLFEYKFVNSRLINSTFLHNKEGCPLDVTGTGEGAYMVY;Sequence ID 10), the region indicated by the amino acid sequence from positions 648 to 651 (sequence ID 11), and the region indicated by the amino acid sequence from positions 709 to 712 (sequence ID 12). In addition, the intravesicular domain includes, for example, the region indicated by a subsequence of any length in Sequence IDs 7 to 12. The length of the subsequence is the same as that described for the anti-SYT2 N-terminal antibody. More specifically, for example, this includes the region indicated by the amino acid sequence from positions 451 to 550 of SEQ ID NO: 6 (SEQ ID NO: 13:MGVWFTMSFSYYGLTVWFPDMIRHLQAVDYASRTKVFPGERVEHVTFNFTLENQIHRGGQYFNDKFIGLRLKSVSFEDSLFEECYFEDVTSSNTFFRNCT) and the protein region of synaptic vesicle glycoprotein 2A having a mutant amino acid sequence in which one or more amino acids are substituted, inserted, deleted, and / or added to the amino acid sequence of SEQ ID NO: 13.

[0034] Synaptogyrin 1 (SYNGR1) refers to one of the four-transmembrane proteins belonging to the synaptogyrin family. This protein is present in the presynaptic vesicles of nerve cells and is thought to be involved in regulatory exocytosis, synaptic vesicle formation and maturation, and synaptic plasticity. An example of synaptogyrin 1 is a human-derived protein consisting of 233 amino acids, represented by the amino acid sequence SEQ ID NO: 14.

[0035] In this specification, "intravesical domain of synaptogyrin 1" refers to all or part of the intravesical domain of synaptogyrin 1. The entire intravesical domain includes, for example, the region indicated by the amino acid sequence from positions 45 to 71 in SEQ ID NO: 14 (sequence: NEGYLNSASEGEEFCIYNRNPNACSYG; SEQ ID NO: 15) and the region indicated by the amino acid sequence from positions 125 to 148 (sequence: YLANQWQVSKPKDNPLNEGTDAAR; SEQ ID NO: 16). Part of the intravesical domain includes, for example, the region indicated by a subsequence of any length in SEQ ID NOs. 15 and 16. The length of the subsequence is as described for the anti-SYT2 N-terminal antibody. More specifically, for example, the protein region of synaptogyrin 1 includes the region indicated by the amino acid sequence from positions 130 to 146 of SEQ ID NO: 14 (SEQ ID NO: 17: WQVSKPKDNPLNEGTDA) and a mutant amino acid sequence in which one or more amino acids are substituted, inserted, deleted, and / or added to the amino acid sequence of SEQ ID NO: 14.

[0036] Synaptophysin (SYP) refers to one of the four-transmembrane proteins belonging to the synaptophysin / synaptobrevin family. This protein is thought to be involved in organizing vesicle membrane components, targeting vesicles to the cell membrane, and regulating 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 consisting of 313 amino acids, represented by the amino acid sequence SEQ ID NO: 18.

[0037] In this specification, "intravesical domain of synaptophysin" refers to all or part of the intravesical domain of synaptophysin. The entire intravesical domain includes, for example, the region indicated by the amino acid sequence from positions 50 to 106 in SEQ ID NO: 18 (sequence: ELQLSVDCANKTESDLSIEVEFEYPFRLHQVYFDAPTCRGGTTKVFLVGDYSSSAEF; SEQ ID NO: 19) and the region indicated by the amino acid sequence from positions 162 to 199 (sequence: KGLSDVKMATDPENIIKEMPVCRQTGNTCKELRDPVTS; SEQ ID NO: 20). Part of the intravesical domain includes, for example, the region indicated by a subsequence of any length in SEQ ID NOs: 19 and 20. The length of the subsequence is as described for the anti-SYT2 N-terminal antibody. More specifically, for example, this includes the protein region of synaptophysin having a mutant amino acid sequence in which one or more amino acids are substituted, inserted, deleted, and / or added in the amino acid sequence of SEQ ID NO: 18 (SEQ ID NO: 21: CRQTGNTCKELRD) and the amino acid sequence of SEQ ID NO: 21.

[0038] Synaptotagmin 1 (SYT1) refers to one of the single-pass transmembrane proteins belonging to the synaptotagmin family. This protein is thought to be one of the proteins that promote the fusion of synaptic vesicles and the cell membrane in a calcium ion-dependent manner. Synaptotagmin 1 is also known by other names such as P65, SVP65, SYT, and BAGOS. An example of synaptotagmin 1 is a human-derived protein consisting of 422 amino acids, represented by the amino acid sequence SEQ ID NO: 22.

[0039] In this specification, "intravesical domain of synaptotagmin 1" refers to all or part of the intravesical domain of synaptotagmin 1. The entire intravesical domain includes, for example, the region indicated by the amino acid sequence from position 1 to 57 in SEQ ID NO: 22 (sequence: MVSESHHEALAAPPVTTVATVLPSNATEPASPGEGKEDAFSKLKEKFMNELHKIPLP; SEQ ID NO: 23). Part of the intravesical domain includes, for example, the region indicated by a subsequence of any length in SEQ ID NO: 23. The length of the subsequence is as described for the anti-SYT2 N-terminal antibody. More specifically, it includes, for example, the region indicated by the amino acid sequence from position 1 to 8 in SEQ ID NO: 22 (sequence ID NO: MVSASRPE) and the N-terminal portion of synaptotagmin 1 having a mutant amino acid sequence in which one or more amino acids are substituted, inserted, deleted, and / or added in the amino acid sequence of SEQ ID NO: 24.

[0040] Synaptogyrin 3 (SYNGR3) refers to one of the four-transmembrane proteins belonging to the synaptogyrin family. This protein is thought to be involved in regulatory exocytosis, dopamine recycling, and other processes. An example of synaptogyrin 3 is a human-derived protein consisting of 229 amino acids, represented by the amino acid sequence SEQ ID NO: 25.

[0041] In this specification, "intravesical domain of synaptogyrin 3" refers to all or part of the intravesical domain of synaptogyrin 3. The entire intravesical domain includes, for example, the region indicated by the amino acid sequence from positions 51 to 69 in SEQ ID NO: 25 (sequence: TDSGPELRCVFNGNAGACR; SEQ ID NO: 26) and the region indicated by the amino acid sequence from positions 126 to 147 (sequence: LTNQWQRTAPGPATTQAGDAAR; SEQ ID NO: 27). Part of the intravesical domain includes, for example, the region indicated by a subsequence of any length in SEQ ID NOs. 26 and 27. The length of the subsequence is as described for the anti-SYT2 N-terminal antibody.

[0042] The "Vesicular Acetylcholine Transporter (VAChT)" 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 to secretory vesicles and releases it outside the cell. The vesicular acetylcholine transporter is also known by other names such as VACHT, SLC18A3, and CMS21. Specifically, an exemplary vesicular acetylcholine transporter is a human-derived protein consisting of 532 amino acids, represented by the amino acid sequence SEQ ID NO: 29.

[0043] In this specification, "intravesicular domain of vesicular acetylcholine transporter" refers to all or part of the intravesicular domain of vesicular acetylcholine transporter. The entire intravesicular domain includes, for example, the region indicated by the amino acid sequence from positions 55 to 125 in SEQ ID NO: 29 (sequence: PIVPDYIAHMRGGGEGPTRTPEVWEPTLPLPTPANASAYTANTSASPTAAWPAGSALRPRYPTESEDVKIG; SEQ ID NO: 30), the region indicated by the amino acid sequence from positions 174 to 182 (sequence: DYATLFAAR; SEQ ID NO: 31), the region indicated by the amino acid sequence from positions 235 to 242 (sequence: LYEFAGKR; SEQ ID NO: 32), the region indicated by the amino acid sequence from positions 311 to 325 (sequence: TIATWMKHTMAASEW; SEQ ID NO: 33), the region indicated by the amino acid sequence from positions 378 to 388 (sequence: RSFAPLVVSLC; SEQ ID NO: 34), and the region indicated by the amino acid sequence from positions 444 to 447 (sequence: LGPI; SEQ ID NO: 35). Furthermore, the intravesicular domain includes, for example, the region represented by a subsequence of any length in SEQ ID NOs. 30-35. The length of the subsequence is as described for the anti-SYT2 N-terminal antibody.

[0044] High Affinity Choline Transporter 1 (CHT1) refers to one of the 13 transmembrane proteins belonging to Solute Carrier Family 5. This protein is a sodium- and chloride-dependent transmembrane transporter that takes up choline from outside the cell with high affinity for acetylcholine synthesis. High affinity choline transporters are also known by other names such as SLC5A7, HCHT, and CHT. An exemplary high affinity choline transporter is a human-derived protein consisting of 580 amino acids, represented by the amino acid sequence SEQ ID NO: 36.

[0045] In this specification, "intravesical domain of high affinity choline transporter 1" refers to all or part of the intravesical domain of high affinity choline transporter 1. The entire intravesical domain includes, for example, the region indicated by the amino acid sequence from position 1 to 6 in SEQ ID NO: 36 (sequence: MAFHVE; SEQ ID NO: 37), This region includes the amino acid sequence from positions 70 to 81 (sequence: GTAEAVYVPGYG; SEQ ID NO: 38), the amino acid sequence from positions 147 to 164 (sequence: GEMFWAAAIFSALGATISVIIDVDMHIS; SEQ ID NO: 39), the amino acid sequence from positions 213 to 237 (sequence: ADIGFTAVHAKYQKPWLGTVDSSEV; SEQ ID NO: 40), the amino acid sequence from positions 296 to 317 (sequence: ASTDWNQTAYGLPDPKTTEEAD; SEQ ID NO: 41), the amino acid sequence from positions 398 to 406 (sequence: LTKTVYGLW; SEQ ID NO: 42), and the amino acid sequence from positions 457 to 481 (sequence: QPLIFYPGYYPDDNGIYNQKFPFKT; SEQ ID NO: 43). Furthermore, the intravesicular domain includes, for example, the region represented by a subsequence of any length in SEQ ID NOs. 37-43. The length of the subsequence is as described for the anti-SYT2 N-terminal antibody.

[0046] Vesicular Glutamate Transporter 1 (VGLUT1) refers to one of the 12-transmembrane proteins belonging to the vesicular glutamate transporter family. This protein is considered a multifunctional cotransport transporter that transports multiple types of ions, including sodium, phosphate, L-glutamate, and chloride ions. Vesicular glutamate transporter 1 is also known by other names such as SLC17A7 and BNPI. An exemplary vesicular glutamate transporter 1 is a human-derived protein consisting of 560 amino acids, represented by the amino acid sequence number 44.

[0047] In this specification, "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 indicated by the amino acid sequence from positions 85 to 116 in SEQ ID NO: 44 (sequence: VAIVSMVNNSTTHRGGHVVVQKAQFSWDPETV; SEQ ID NO: 45), the region indicated by the amino acid sequence from positions 162 to 169 (sequence: PSAARVHY; SEQ ID NO: 46), the region indicated by the amino acid sequence from positions 230 to 236 (sequence: QYSGWSS; SEQ ID NO: 47), the region indicated by the amino acid sequence from positions 324 to 341 (sequence: SQPAYFEEVFGFEISKVG; SEQ ID NO: 48), the region indicated by the amino acid sequence from positions 400 to 401 (sequence: SK), and the region indicated by the amino acid sequence from positions 457 to 469 (sequence: GAMTKHKTREEWQ; SEQ ID NO: 49). In addition, the intravesicular domain includes, for example, the region indicated by a subsequence of any length in SEQ ID NOs: 45 to 49. The length of the sub-sequence is the same as that described for the anti-SYT2 N-terminal antibody.

[0048] Vesicular glutamate transporter 3 (VGLUT3) refers to one of the 10-transmembrane proteins belonging to the vesicular glutamate transporter family. This protein is considered a multifunctional uniporter that transports multiple types of ions, including sodium, phosphate, L-glutamate, and chloride ions. Vesicular glutamate transporter 3 is also known by other names such as SLC17A8 and DFNA25. An exemplary vesicular glutamate transporter 3 is a human-derived protein consisting of 589 amino acids, represented by the amino acid sequence number 50.

[0049] In this specification, "intravesical domain of vesicular glutamate transporter 3" refers to all or part of the intravesical domain of vesicular glutamate transporter 3. The entire intravesicular domain includes, for example, the region indicated by the amino acid sequence from positions 98 to 130 in SEQ ID NO: 50 (sequence: VAIVEMVNNSTVYVDGKPEIQTAQFNWDPETVG; SEQ ID NO: 51), the region indicated by the amino acid sequence from positions 175 to 182 (sequence: PSAARVHY; SEQ ID NO: 52), the region indicated by the amino acid sequence from positions 243 to 249 (sequence: QYIGWSS; SEQ ID NO: 53), the region indicated by the amino acid sequence from positions 336 to 353 (sequence: SQPAYFEEVFGFAISKVG; SEQ ID NO: 54), the region indicated by the amino acid sequence from positions 412 to 413 (sequence: TK), and the region indicated by the amino acid sequence from positions 469 to 481 (sequence: GAMTRHKTREEWQ; SEQ ID NO: 55). In addition, the intravesicular domain includes, for example, the region indicated by a subsequence of any length in SEQ ID NOs: 51 to 55. The length of the sub-sequence is the same as that described for the anti-SYT2 N-terminal antibody.

[0050] The term "Vesicular GABA Transporter (VGAT)" refers to a 10-transmembrane protein belonging to the amino acid / polyamine transporter family. This protein is an antiporter that transports 4-aminobutanoic acid or glycine from the cytoplasm into vesicles by exchanging it with protons within the vesicle for secretion from nerve endings. Vesicular GABA transporters are also known by other names such as VIAAT, SLC32A1, and BA122O1.1. An exemplary vesicular GABA transporter is a human-derived protein consisting of 525 amino acids, represented by the amino acid sequence number 56.

[0051] In this specification, "intravesical domain of vesicular GABA transporter" refers to all or part of the intravesical domain of vesicular GABA transporter. The entire intravesical domain includes, for example, the region indicated by the amino acid sequence from positions 154 to 204 in SEQ ID NO: 56 (sequence: FAAVVCCYTGKILIACLYEENEDGEVVRVRDSYVAIANACCAPRFPTLGGR; SEQ ID NO: 57), the region indicated by the amino acid sequence from positions 287 to 305 (sequence: SRARDWAWEKVKFYIDVKK; SEQ ID NO: 58), the region indicated by the amino acid sequence from positions 363 to 383 (sequence: ADETKEVITDNLPGSIRAVVN; SEQ ID NO: 59), the region indicated by the amino acid sequence from positions 460 to 461 (sequence: TG), and the region indicated by the amino acid sequence from positions 511 to 525 (sequence: SLEGLIEAYRTNAED; SEQ ID NO: 60). Furthermore, the intravesicular domain includes, for example, the region represented by a subsequence of any length in SEQ ID NOs. 57-60. The length of the subsequence is as described for the anti-SYT2 N-terminal antibody.

[0052] Synaptic vesicle glycoprotein 2B (SV2B) 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 regulation of regulated secretion in nerve cells and endocrine cells, and is suggested to function as a protein receptor for botulinum neurotoxin E in nerve cells. Synaptic vesicle glycoprotein 2B is also known by other names such as KIAA0735, HsT19680, and SLC22B2. An example of synaptic vesicle glycoprotein 2B is a human-derived protein consisting of 683 amino acids, represented by the amino acid sequence SEQ ID NO: 61.

[0053] In this specification, "intravesical domain of synaptic vesicle glycoprotein 2B" refers to all or part of the intravesical domain of synaptic vesicle glycoprotein 2B. The entire intravesical domain includes, for example, the region indicated by the amino acid sequence from positions 130 to 148 in SEQ ID NO: 61 (sequence: SFALPSAEKDMCLSSSKKG; SEQ ID NO: 62), the region indicated by the amino acid sequence from positions 204 to 205 (sequence: CR), the region indicated by the amino acid sequence from positions 259 to 277 (sequence: PHYGWGFSMGTNYHFHSWR; SEQ ID NO: 63), and the amino acid sequence from positions 412 to 535 (sequence: PDMIRYFQDEEYKSKM This includes the region indicated by KVFFGEHVYGATINFTMENQIHQHGKLVNDKFTRMYFKHVLFEDTFFDECYFEDVTSTDTYFKNCTIESTIFYNTDLYEHKFINCRFINSTFLEQKEGCHMDLEQDND (Sequence ID 64), the region indicated by the amino acid sequence from positions 587 to 592 (sequence ID: NSESAM; Sequence ID 65), and the region indicated by the amino acid sequence from positions 650 to 653 (sequence ID: GITK; Sequence ID 66). In addition, as part of the intravesicular domain, it includes, for example, the region indicated by a subsequence of any length in Sequence IDs 62 to 66. The length of the subsequence is in accordance with the description for the anti-SYT2 N-terminal antibody.

[0054] Synaptic vesicle glycoprotein 2C (SV2C) 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 regulation of regulated secretion in nerve cells and endocrine cells, and is involved in neurotransmitter transport and transmembrane transport. Synaptic vesicle glycoprotein 2C is also known by other names such as SLC22B3 and KIAA1054. An example of synaptic vesicle glycoprotein 2C is a human-derived protein consisting of 727 amino acids, represented by the amino acid sequence SEQ ID NO: 67.

[0055] In this specification, "intravesical domain of synaptic vesicle glycoprotein 2C" refers to all or part of the intravesical domain of synaptic vesicle glycoprotein 2C. The entire intravesical domain includes, for example, the region indicated by the amino acid sequence from positions 176 to 191 in SEQ ID NO: 67 (sequence: LPSAETDLCIPNSGSG; SEQ ID NO: 68), the region indicated by the amino acid sequence at position 248 (sequence: R), the region indicated by the amino acid sequence from positions 302 to 320 (sequence: PHYGWSFSMGSAYQFHSWR; SEQ ID NO: 69), and the amino acid sequence from positions 459 to 578 (sequence: KPLQSDEYALLTRNVERDKYA This includes the region indicated by NFTINFTMENQIHTGMEYDNGRFIGVKFKSVTFKDSVFKSCTFEDVTSVNTYFKNCTFIDTVFDNTDFEPYKFIDSEFKNCSFFHNKTGCQITFDDDYS (Sequence ID 70), the region indicated by the amino acid sequence from positions 631 to 636 (sequence ID 71), and the region indicated by the amino acid sequence from positions 691 to 698 (sequence ID 72). In addition, as part of the intravesicular domain, it includes the region indicated by a subsequence of any length in sequences 68 to 72, for example. The length of the subsequence is in accordance with the description for the anti-SYT2 N-terminal antibody.

[0056] Vesicle-associated membrane protein 1 (VAMP1) refers to one of the single-pass transmembrane proteins belonging to the synaptobrevin family. This protein is thought to be involved in the targeting of transport vesicles to the cell membrane and / or membrane fusion. Vesicle-associated membrane protein 1 is also known by other names such as SYB1, CMS25, SPAX1, and synaptobrevin 1. An exemplary example of vesicle-associated membrane protein 1 is a human-derived protein consisting of 118 amino acids, represented by the amino acid sequence number 73.

[0057] In this specification, "intravesical domain of vesicle-associated membrane protein 1" refers to all or part of the intravesical domain of vesicle-associated membrane protein 1. The entire intravesical domain includes, for example, the region indicated by the amino acid sequence (sequence: FT) from positions 117 to 118 in SEQ ID NO: 73.

[0058] Synaptogyrin 4 (SYNGR4) 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 example of synaptogyrin 4 is a human-derived protein consisting of 234 amino acids, represented by the amino acid sequence SEQ ID NO: 74.

[0059] In this specification, "intravesical domain of synaptogyrin 4" refers to all or part of the intravesical domain of synaptogyrin 4. The entire intravesical domain includes, for example, the region indicated by the amino acid sequence from positions 46 to 65 in SEQ ID NO: 74 (sequence: YQNKMESPQLHCILNSNSVA; SEQ ID NO: 75) and the region indicated by the amino acid sequence from positions 125 to 144 (sequence: ANQWQHSPPKEFLLGSSSAQ; SEQ ID NO: 76). Part of the intravesical domain includes, for example, the region indicated by a subsequence of any length in SEQ ID NOs. 75 and 76. The length of the subsequence is as described for the anti-SYT2 N-terminal antibody.

[0060] Synaptotagmin 4 (SYT4) refers to one of the single-pass 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. An example of synaptotagmin 4 is a human-derived protein consisting of 425 amino acids, represented by the amino acid sequence number 77.

[0061] In this specification, "intravesical domain of synaptotagmin 4" refers to all or part of the intravesical domain of synaptotagmin 4. The entire intravesical domain includes, for example, the region indicated by the amino acid sequence from position 1 to 16 in SEQ ID NO: 77 (sequence: MAPITTSREEFDEIPT; SEQ ID NO: 78). Part of the intravesical domain includes, for example, the region indicated by a subsequence of any length in SEQ ID NO: 78. The length of the subsequence is as described for the anti-SYT2 N-terminal antibody.

[0062] Synaptotagmin 7 (SYT7) refers to one of the single-pass transmembrane proteins belonging to the synaptotagmin family. This protein is thought to be one of the proteins that promotes the fusion of secretory vesicles and synaptic vesicles with the cell membrane in a calcium ion-dependent manner. Synaptotagmin 7 is also known by other names such as IPCA-7, PCANAP7, and MGC150517. An example of synaptotagmin 7 is a human-derived protein consisting of 403 amino acids, represented by the amino acid sequence number 79.

[0063] In this specification, "intravesical domain of synaptotagmin 7" refers to all or part of the intravesical domain of synaptotagmin 7. The entire intravesical domain includes, for example, the region indicated by the amino acid sequence from position 1 to 16 in SEQ ID NO: 79 (sequence: MYRDPEAASPGAPSRD; SEQ ID NO: 80). Part of the intravesical domain includes, for example, the region indicated by a subsequence of any length in SEQ ID NO: 80. The length of the subsequence is as described for the anti-SYT2 N-terminal antibody.

[0064] Secretory Carrier Membrane Protein 5 (SCAMP5) refers to one of the four-transmembrane proteins belonging to the secretory carrier membrane protein family. This protein is thought to be involved in calcium-dependent exocytosis of cytokines and other substances. Secretory carrier membrane protein 5 is also known by other names such as MGC24969 and HSCAMP5. An exemplary secretory carrier membrane protein 5 is a human-derived protein consisting of 235 amino acids, represented by the amino acid sequence number 81.

[0065] In this specification, "intravesical domain of secreted carrier-associated membrane protein 5" refers to all or part of the intravesical domain of secreted carrier-associated membrane protein 5. The entire intravesical domain includes, for example, the region indicated by the amino acid sequence from positions 61 to 67 in SEQ ID NO: 81 (sequence: WLIGGGG; SEQ ID NO: 82) and the region indicated by the amino acid sequence from positions 126 to 148 (sequence: IPGWGVCGWIATISFFGTNIGSA; SEQ ID NO: 83). Part of the intravesical domain includes, for example, the region indicated by a partial sequence of any length in SEQ ID NOs: 82 and 83. The length of the partial sequence is as described for the anti-SYT2 N-terminal antibody.

[0066] Synaptic Vesicle 2-Related Protein (SVOP) refers to one of the 12-transmembrane proteins belonging to the synaptic vesicle glycoprotein 2 family, a major facilitator superfamily. This protein is thought to possess transmembrane transporter activity. Synaptic vesicle glycoprotein 2-related proteins are also known by other names such as SLC22B4, DKFZp761H039, and SCF22B4. An exemplary synaptic vesicle glycoprotein 2-related protein is a human-derived protein consisting of 548 amino acids, represented by the amino acid sequence number 84.

[0067] In this specification, "intravesicular domain of synaptic vesicle glycoprotein 2-related protein" refers to all or part of the intravesicular domain of synaptic vesicle glycoprotein 2-related protein. The entire intravesicular domain includes, for example, the region indicated by the amino acid sequence from positions 109 to 122 in SEQ ID NO: 84 (sequence: PQLHCEWRLPSWQV; SEQ ID NO: 85), the region indicated by the amino acid sequence from positions 178 to 180 (sequence: VLR), the region indicated by the amino acid sequence from positions 231 to 238 (sequence: VMPSLGWR; SEQ ID NO: 86), the region indicated by the amino acid sequence from positions 338 to 373 (sequence: TTELFQAGDVCGISSRKKAVEAKCSLACEYLSEEDY; SEQ ID NO: 87), the region indicated by the amino acid sequence from positions 423 to 424 (sequence: RN), and the region indicated by the amino acid sequence from positions 479 to 489 (sequence: AQVMLESSVYL; SEQ ID NO: 88). In addition, the intravesicular domain includes, for example, the region indicated by a subsequence of any length in SEQ ID NOs: 85 to 88. The length of the sub-sequence is the same as that described for the anti-SYT2 N-terminal antibody.

[0068] Excitatory Amino Acid Transporter 3 (EAAT3) refers to one of the eight-transmembrane proteins belonging to the glutamate transporter subfamily. This protein is thought to be a sodium-dependent, high-affinity amino acid transporter that mediates the uptake of glutamate, aspartate, and cysteine. Excitatory Amino Acid Transporter 3 is also known by other names such as SLC1A1, HEAAC1, and EAAC1. An exemplary Excitatory Amino Acid Transporter 3 is a human-derived protein consisting of 524 amino acids, represented by the amino acid sequence number 89.

[0069] In this specification, "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 indicated by the amino acid sequence from positions 39 to 61 in SEQ ID NO: 89 (sequence: REHSNLSTLEKFYFAFPGEILMR; SEQ ID NO: 90), the region indicated by the amino acid sequence from positions 115 to 205 (sequence: SIKPGVTQKVGEIARTGSTPEVSTVDAMLDLIRNMFPENLVQACFQQYKTKREEVKPPSDPEMNMTEESFTAVMTTAISKNKTKEYKIVGM; SEQ ID NO: 91), the region indicated by the amino acid sequence from positions 267 to 286 (sequence: AGKIIEVEDWEIFRKLGLYM; SEQ ID NO: 92), the region indicated by the amino acid sequence from positions 381 to 393 (sequence: IAQLNDLDLGIGQ; SEQ ID NO: 93), and the region indicated by the amino acid sequence from positions 428 to 440 (sequence: LPAEDVTLIIAVD; SEQ ID NO: 94). Furthermore, the intravesicular domain includes, for example, the region indicated by a subsequence of any length in SEQ ID NOs. 90-94. The length of the subsequence is as described for the anti-SYT2 N-terminal antibody.

[0070] Autophagy-Related Protein 9A (ATG9A) refers to one of the five-transmembrane proteins belonging to the Autophagy-Related Protein 9 family. This protein is a phospholipid scramblase involved 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. An exemplary Autophagy-Related Protein 9A is a human-derived protein consisting of 839 amino acids, represented by the amino acid sequence number 95.

[0071] In this specification, "intravesical domain of autophagy-related protein 9A" refers to all or part of the intravesical domain of autophagy-related protein 9A. The entire intravesical domain includes, for example, the region indicated by the amino acid sequence from positions 85 to 128 in SEQ ID NO: 95 (sequence: SCVDYDILFANKMVNHSLHPTEPVKVTLPDAFLPAQVCSARIQE; SEQ ID NO: 96) and the region indicated by the amino acid sequence from positions 398 to 406 (sequence: DEDVLAVEH; SEQ ID NO: 97). Part of the intravesical domain includes, for example, the region indicated by a subsequence of any length in SEQ ID NOs. 96 and 97. The length of the subsequence is as described for the anti-SYT2 N-terminal antibody.

[0072] Glucose transporter type 4 (GLUT4) refers to one of the 12-transmembrane proteins belonging to the sugar transporter family. This protein is an insulin-regulated glucose transporter and plays a crucial role in removing glucose from the systemic circulation. Glucose transporter type 4 is also known by other names such as SLC2A4. An exemplary glucose transporter type 4 is a human-derived protein consisting of 509 amino acids, represented by the amino acid sequence number 98.

[0073] In this specification, "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 indicated by the amino acid sequence from positions 46 to 81 in SEQ ID NO: 98 (sequence: NAPQKVIEQSYNETWLGRQGPEGPSSIPPGTLTTLW; SEQ ID NO: 99), the region indicated by the amino acid sequence from positions 133 to 142 (sequence: ASYEMLILGR; SEQ ID NO: 100), the region indicated by the amino acid sequence from positions 193 to 201 (sequence: ESLLGTASL; SEQ ID NO: 101), the region indicated by the amino acid sequence from positions 309 to 323 (sequence: YSTSIFETAGVGQPA; SEQ ID NO: 102), the region indicated by the amino acid sequence from positions 375 to 384 (sequence: ERVPAMSYVS; SEQ ID NO: 103), and the region indicated by the amino acid sequence from positions 439 to 445 (sequence: QYVAEAM; SEQ ID NO: 104). Furthermore, the intravesicular domain includes, for example, the region represented by a subsequence of any length in sequence numbers 99-104. The length of the subsequence is as described for the anti-SYT2 N-terminal antibody.

[0074] ATPase H+ Transporting Accessory Protein 1 (ATP6AP1) refers to one of the single-pass 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 necessary for acidifying the lumen of secretory vesicles. ATPase H+ Transporting Accessory Protein 1 is also known by other names such as VATPS1, XAP3, ATP6IP1, and ATP6S1. An example of ATPase H+ Transporting Accessory Protein 1 is a human-derived protein consisting of 470 amino acids, represented by the amino acid sequence SEQ ID NO: 105.

[0075] In this specification, "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 419 in SEQ ID NO: 105 (sequence: EQQVPLVLWSSDRDLWAPAADTHEGHITSDLQLSTYLDPALELGPRNVLLFLQDKLSIEDFTAYGGVFGNKQDSAFSNLENALDLAPSSLVLPAVDWYAVSTLTTYLQEKLGASPLHVDLATLRELKLNASLPALLLIRLPYTASSGLMAPREVLTGNDEVIGQVLSTLKSEDV The region indicated by PYTAALTAVRPSRVARDVAVVAGGLGRQLLQKQPVSPVIHPPVSYNDTAPRILFWAQNFSVAYKDQWEDLTPLTFGVQELNLTGSFWNDSFARLSLTYERLFGTTVTFKFILANRLYPVSARHWFTMERLEVHSNGSVAYFNASQVTGPSIYSFHCEYVSSLSKKGSLLVARTQPSPWQMMLQDFQIQAFNVMGEQFSYASDCA;Sequence ID 106) is included. Additionally, the intravesicular domain includes, for example, the region indicated by a subsequence of any length in Sequence ID 106. The length of the subsequence is in accordance with the description for the anti-SYT2 N-terminal antibody.

[0076] Furthermore, the targeting agent of the present invention can use an antibody capable of binding to the non-protein region of membrane proteins present in vesicles that is exposed to the vesicular lumen. Examples of such non-protein regions include glycans and lipids. In addition to antibodies capable of binding to glycans on intravesicular domains as described above, antibodies capable of binding to a portion of the lipid anchor of a lipid-modified protein that does not have a transmembrane domain, if that portion is exposed to the vesicular lumen, can be used. Specifically, for example, an antibody capable of binding to the lipid anchor portion of Ras-related proteins such as Rab3a (SEQ ID NO: 110) can be used. Also, for example, an antibody capable of binding to the hydrophobic domain present in the vesicular membrane of superficial membrane proteins can be used. For example, an antibody capable of binding to the hydrophobic domain present in the vesicular membrane of synapsin family proteins such as synapsin 1 (SYN1: SEQ ID NO: 107), synapsin 2 (SYN2: SEQ ID NO: 108), and synapsin 3 (SYN3: SEQ ID NO: 109) can be used.

[0077] The animals from which the membrane proteins originate in this specification are not particularly limited, but may be derived from various vertebrates and mammals, as described later with respect to cells, and preferably from humans.

[0078] In this specification, "targeting agent" refers to a drug used to deliver a specific substance to a target. In this specification, by using a targeting agent, a desired substance (labeled substance and / or bioactive substance) is transported to motor neurons, particularly motor neuron synapses. Furthermore, in targeting of synapses and synaptic vesicles, the substance is taken up into the cell, particularly into the synaptic vesicles, by endocytosis and delivered to the cell body. Moreover, the targeting agent of the present invention allows the transported substance to exert bioactivity in the cytoplasm.

[0079] For example, the targeting agent of the present invention may be taken up into synaptic vesicles, and the transported desired substance (labeled substance and / or bioactive substance) may permeate the synaptic vesicle membrane and move into the cytoplasm. Specifically, for example, when a bioactive substance is used, at least one bioactive substance in the targeting agent may move into the cytoplasm and act on a biomolecule in the nucleus, a desired biomolecule in the cytoplasm, or a biomolecule on the cytoplasmic membrane. The biomolecule on which the bioactive substance acts is not particularly limited as long as it can be present on the cell membrane or inside the cell, and may be any of the following: high molecular weight compounds such as proteins and nucleic acids, low molecular weight compounds such as lipids, sugars, amino acids, and nucleotides, or ions or atoms such as metal ions.

[0080] Specifically, examples of biomolecules within the nucleus include DNA, RNA (mRNA, siRNA, miRNA, etc.), transcription factors, and nuclear receptors. Examples of biomolecules within the cytoplasm include the nucleic acids mentioned above, as well as the cytoskeleton, enzymes, and metal ions. Examples of biomolecules on the cell membrane include cell membrane lipids, cell membrane receptors, receptor-coupled enzymes, and cell adhesion molecules.

[0081] In this specification, "motor nerve cells" refers to a group of nerve cells that transmit stimuli from the central nervous system to skeletal muscles, which are effectors. Motor nerve cells generally include primary motor nerve cells, which are central nervous system cells, and secondary motor nerve cells, which are peripheral nervous system cells, but in this specification, motor nerve cells refer to secondary motor nerve cells.

[0082] In this specification, "secondary motor neurons" refer to motor neurons that have their cell bodies in the anterior horn of the spinal cord or the brainstem and extend their axons to the junction with skeletal muscles. Examples of motor neurons in this specification include α motor neurons, β motor neurons, and γ motor neurons. In addition to spinal nerves that have their 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. Motor neurons in this specification usually secrete acetylcholine as a neurotransmitter and are classified as cholinergic neurons. However, motor neurons that secrete neurotransmitters other than acetylcholine may also be included. The muscle cells to which motor neurons project in this specification are skeletal muscle cells.

[0083] In this specification, "skeletal muscle cells" refers to cells that constitute striated muscle, which moves the skeleton, or cells that have the phenotype thereof. Skeletal muscle cells in this specification broadly include muscle cells attached to bone and other muscle cells contained in skeletal muscle, such as muscle spindles. The types of skeletal muscle are not particularly limited, but examples include the diaphragm, vastus lateralis, vastus medialis, rectus femoris, vastus intermedius, biceps brachii, tibialis anterior, tibialis posterior, gastrocnemius, soleus, deltoid, latissimus dorsi, sternocleidomastoid, intercostal muscles, ocular muscles, facial muscles, tongue muscles, stapedius muscles, etc. Furthermore, skeletal muscle cells in this specification also include cultured skeletal muscle cells such as cells differentiated in vitro from artificial stem cells (iPS cells and ES cells, etc.) and / or natural stem cells (mesenchymal stem cells and skeletal muscle stem cells, etc.).

[0084] In this specification, cells may be derived from vertebrates. Vertebrates include fish, reptiles, amphibians, birds, and mammals. Specific mammals include, for example, primates (e.g., humans). Cells may also be derived from livestock (chickens, horses, cattle, sheep, goats, pigs, etc.), pets (tropical fish, lizards, dogs, cats, rabbits, etc.), and laboratory animals (frogs, mice, rats, monkeys, etc.). Cells do not need to be derived from a single type of tissue, individual, or animal species, but may be a mixture of multiple types of cells. Furthermore, the health status of the tissue and individual from which the cells originate is not particularly limited.

[0085] According to the targeting agent of the present invention, a desired substance (labeled substance and / or bioactive substance) can be targeted to motor neurons (e.g., axon terminals, axons, axonal papillae, cell bodies, dendrites, etc. of motor neurons) via motor neuron synapses.

[0086] In this specification, "synapse" refers to a junction, including a gap, formed between the axon terminal of one nerve cell and the dendrite of another nerve cell (in the case of the central nervous system) or cells of skeletal muscle, organs, etc. (in the case of the peripheral nervous system).

[0087] In this specification, a synapse may be a chemical synapse, such as an excitatory synapse or an inhibitory synapse. In this specification, a synapse may be a synapse formed between one nerve cell and another nerve cell (for example, a synapse formed between the axon of one nerve cell and the dendrite of another nerve cell), or a synapse formed between a nerve cell and another type of cell (such as a muscle cell), but preferably a synapse formed by the presynaptic portion of a nerve cell and the postsynaptic portion on a skeletal muscle cell (also referred to as a "neuromuscular junction").

[0088] In this specification, "presynaptic region" refers to the enlarged portion formed at the axon terminal of a nerve cell at a synapse, and "postsynaptic region" refers to the portion of another nerve cell's dendrite or other cell such as skeletal muscle or organ that faces the presynaptic region. Furthermore, "synaptic cleft" refers to the space between the presynaptic and postsynaptic regions. At a synapse, neurotransmitters accumulated in synaptic vesicles present in the presynaptic region are released into the synaptic cleft and transmit signals by binding to receptors present in the postsynaptic region.

[0089] In this specification, "synaptic vesicle" refers to a secretory vesicle present in the cytoplasm of a presynaptic nerve cell. In this specification, synaptic vesicles include not only vesicles that contain neurotransmitters and fuse with the cell membrane in response to stimuli to release neurotransmitters into the synaptic cleft, but also vesicles that have been recovered into the nerve cell by endocytosis (including bulk endocytosis) after the release of neurotransmitters.

[0090] 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, is taken up into synaptic vesicles by endocytosis, and can then be delivered to the cell body of motor neurons. Therefore, the targeting agent of the present invention makes it possible to target a desired substance (labeled substance and / or bioactive substance) to the inside of motor neurons, particularly to the cell body of motor neurons.

[0091] In this specification, "antibody capable of binding to the intravesicular domain of a membrane protein (intravesicular domain antibody)" refers to an antibody that uses the intravesicular domain of a membrane protein as an antigen and is capable of specifically binding to it.

[0092] For example, in this specification, "antibody capable of binding to the intravesicular domain (N-terminal portion) of synaptotagmin 2 (anti-SYT2 N-terminal antibody)" refers to an antibody that uses the intravesicular domain of synaptotagmin 2 as an antigen and is capable of specifically binding to it.

[0093] Antibodies containing intravesicular domains, such as anti-SYT2 N-terminal antibodies, include both monoclonal and polyclonal antibodies. The antibodies may be IgG antibody molecules, IgM antibody molecules, or their antigen-binding fragments and antigen-binding derivatives. For example, the antibodies may be complete antibodies, Fab, Fab', F(ab')2 fragments, or single-chain antibody (scFv) fragments (scFv-Fc), sc(Fv)2, Fv, diabodies, etc., in which the heavy chain variable region (VH) and light chain variable region (VL) are linked by a linker. Those skilled in the art can easily obtain or synthesize these antibodies using the intravesicular domains of the aforementioned membrane proteins, such as the N-terminal polypeptide of synaptotagmin 2, or other known membrane proteins, as antigens. Commercially available antibodies can also be used in this invention. The antibody may be a human chimeric antibody, a humanized antibody, or a human antibody. When the conjugate or targeting agent of the present invention is administered to a human, the antibody portion is preferably a human chimeric antibody, a humanized antibody, or a human antibody.

[0094] The targeting agent of the present invention may include antibodies capable of binding to multiple types of intravesicular domains. In this case, these multiple types of intravesicular domains may belong to the same membrane protein or to different membrane proteins.

[0095] The antibodies that can be used in the present invention also include derivatives that can be understood by those skilled in the art, to the extent that they do not affect antigen binding, such as derivatives that have been modified to facilitate antibody purification or to enhance stability. In this specification, fragments and derivatives that retain binding affinity to the intravesicular domain of synaptotagmin 2 are intended to be included in "antibody" unless otherwise consistent with the context.

[0096] Instead of antibodies as described herein, any molecule capable of binding to the target molecule can be used as a targeting agent or motor neuron visualization agent of the present invention. Examples of molecules capable of binding to the target molecule include aptamers, cyclic peptides, receptors or ligands of the target molecule, or combinations thereof.

[0097] The targeting agent of the present invention may further include an antibody capable of binding to the intravesicular domain (N-terminal portion) of synaptotagmin 2, as well as an antibody capable of binding to the intravesicular domain of a membrane protein, and a desired substance (labeled substance and / or physiologically active substance).

[0098] In this specification, "labeled substance" refers to a substance that emits a signal that can be detected. Examples of labeled substances include fluorescent molecules, luminescent labeling substances that emit light under specific conditions such as chemiluminescent substances, sound-emitting labeling substances that emit sound waves such as photoacoustic effect probes, and radioactive labeling substances. Examples of fluorescent molecules, though not limited to them, include fluorescent proteins, fluorescein and its derivatives, pyrene and its derivatives, and quantum dots. Examples of chemiluminescent substances include enzymes such as peroxidase (HRP) and alkaline phosphatase (ALP). Examples of radioactive labeling substances include, for example 14 C, 3H, 125 Examples of reagents include those containing I. The photoacoustic effect is a phenomenon in which thermoelastic waves are generated by adiabatic expansion accompanying light absorption, and these thermoelastic waves can be detected as acoustic waves. Examples of photoacoustic effect probes include indocyanine green or its derivatives, curcumin derivatives, or choline derivatives. If the absorbance characteristics of the antibody, labeling substance, and physiologically active substance to be used are known, it is not always necessary to use a labeling substance for the photoacoustic effect; for example, a luminescent labeling substance may be detected based on the photoacoustic effect.

[0099] In this specification, "bioactive substance" refers to a substance that can exert a physiological effect directly or indirectly on a living organism or cells. Examples include low-molecular-weight compounds that can exert a physiological effect on target motor nerve cells, functional medium-sized molecules such as peptides and aptamers, and macromolecules including proteins such as antibodies and enzymes, and biomacromolecules such as nucleic acids such as DNA and RNA. For example, drugs or prodrugs such as synapse formation promoters, synapse maintenance agents, muscle enhancers, or nerve cell function modifiers can be used as bioactive substances.

[0100] "Physiological effects" refer to effects that bring about quantitative and / or qualitative changes in biomolecules such as proteins, DNA, and RNA. As a result of physiological effects, the function and properties of 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, or improvement or prevention of hyperactivity of nerve cells can be obtained.

[0101] When the targeting agent of the present invention contains a desired substance (labeled substance and / or physiologically active substance), the antibody capable of binding to the intravesicular domain of membrane proteins, including an antibody capable of binding to the intravesicular domain of synaptotagmin 2, and the desired substance are included either in a state where they are not covalently linked (for example, in a non-covalently linked state) or in a state where they are covalently linked. When included in a state where they are covalently linked, the antibody capable of binding to the intravesicular domain of membrane proteins, including an antibody capable of binding to the intravesicular domain of synaptotagmin 2, and the desired substance form a conjugate (referred to as "the conjugate of the present invention").

[0102] In this specification, "conjugate" refers to a substance in which two or more molecules are linked by covalent bonds. In particular, the conjugate of the present invention is one in which an antibody capable of binding to the intravesicular domain of a membrane protein is linked to a desired substance (a labeled substance and / or a physiologically active substance). Specifically, for example, an antibody capable of binding to the intravesicular domain of synaptotagmin 2 is linked to a desired substance (a labeled substance and / or a physiologically active substance).

[0103] The covalent and non-covalent bonds between the antibody and the desired substance in the targeting agent of the present invention are not particularly limited as long as the antibody, which can bind to the intravesicular domain of synaptotagmin 2, and the desired substance are linked and can reach the vicinity of motor neurons.

[0104] In this specification, "synapse formation promoter" refers to a drug that has the effect of promoting the formation of the presynaptic and / or postsynaptic regions. Synapse formation promotion includes, for example, enhancing the synaptic junction, such as (i) increasing the surface area and / or volume of the presynaptic and / or postsynaptic regions, (ii) increasing and / or qualitatively changing the amount, density, accumulation rate, accumulation frequency, etc. of proteins specifically expressed in the presynaptic region (e.g., synapsin 1 or synapsin 2), and (iii) increasing and / or qualitatively changing the amount, density, accumulation rate, accumulation frequency, etc. of proteins specifically expressed in the postsynaptic region (e.g., LRRTM family proteins).

[0105] In this specification, “synaptic maintenance agent” refers to a drug that has the effect of suppressing or assisting the retraction of the presynaptic and / or postsynaptic regions. Synaptic maintenance includes, for example, suppressing or assisting the weakening of synaptic junction, for example, (i) suppressing or assisting the decrease in the surface area and / or volume of the presynaptic and / or postsynaptic regions, (ii) suppressing or assisting the decrease and / or qualitative change of the amount, density, accumulation rate, accumulation frequency, etc. of proteins specifically expressed in the presynaptic region (e.g., synapsin 1 or synapsin 2), and (iii) suppressing or assisting the decrease and / or qualitative change of the amount, density, accumulation rate, accumulation frequency, etc. of proteins specifically expressed in the postsynaptic region (e.g., LRRTM family proteins).

[0106] In this specification, "muscle-enhancing agent" refers to a drug that has the effect of increasing muscle mass or inhibiting muscle weakness, or promoting such action. The type of muscle enhancement is not particularly limited as long as muscle function is strengthened, but examples include an increase in the surface area and / or volume of muscle, an increase and / or qualitative change in the number and density of each component constituting muscle such as muscle bundles, muscle fibers, myofibrils, sarcomeres, and muscle cells, and / or a change in the expression level of specific proteins in the cells constituting muscle, an increase in muscle mass and / or muscle strength (e.g., muscle mass or muscle strength of skeletal muscle), or an effect that causes muscle weakness through these actions.

[0107] Specific synapse formation promoters, synapse maintenance agents, and muscle strengthening agents include, but are not limited to, compounds disclosed in Japanese Patent Application Publication No. 2022-053535 (e.g., thiamine and its derivatives) and compounds disclosed in Japanese Patent Application Publication No. 2023-028848 (e.g., atropine, busulfan, chromocarb, procainamide, udenafil, propifenazone and their derivatives), which have been discovered by the present inventors.

[0108] In this specification, "neuronal cell function modifier" refers to a drug that alters or enhances the function exhibited by nerve cells. Modification of nerve cell function is not particularly limited as long as it alters the degree and / or properties of the function of the nerve cell, but includes, for example, changes in the electrophysiological properties of nerve cells (such as the properties of conduction and transmission of stimuli), changes in gene expression patterns, and changes in morphological properties (such as the extension, retraction, and branching of neurites, and the formation and retraction of synapses).

[0109] Specific functional modifiers include, but are not limited to, AP-1 inhibitors (e.g., compounds disclosed in WO2020 / 196725 discovered by the present inventors), FUS inhibitors, SOD1 inhibitors, TDP-43 inhibitors (e.g., compounds of anacardic acid), KIF1A inhibitors, and cytoskeletal modifiers such as monomethyl auristatin E (MMAE).

[0110] In this specification, "cytoskeleton modifier" refers to a drug that inhibits and / or promotes one or more of the cytoskeleton formation, maintenance, degradation, branching, pathway, and localization. Cytoskeleton modifiers in this specification also include drugs that modify cytoskeleton formation, etc., by acting on molecules other than the cytoskeleton. The cytoskeleton includes microtubules, intermediate filaments, and actin filaments. For example, a drug that inhibits cytoskeleton formation and maintenance, specifically, a microtubule polymerization inhibitor, can be used as a cytoskeleton modifier.

[0111] These drugs only need to have an effect on nerve cells; they do not need to have a specific effect on motor nerve cells.

[0112] In the conjugate of the present invention, an intravesicular domain antibody such as an anti-SYT2 N-terminal antibody and a labeled substance and / or a physiologically active substance may be directly linked by covalent bonds, or they may be indirectly linked via a linker or the like.

[0113] If the targeting agent of the present invention does not contain a conjugate, it is preferable that the labeling substance and / or bioactive substance have a portion that can bind to an intravesicular domain antibody, such as an anti-SYT2 N-terminal antibody. Specifically, for example, a labeling substance and / or bioactive substance that is covalently bound to an antibody that can bind to an intravesicular domain antibody, such as an anti-SYT2 N-terminal antibody, can be used. In this case, the definitions of "antibody that can bind to an anti-SYT2 N-terminal antibody" and "antibody that can bind to an intravesicular domain antibody" are the same as those described in the definitions of "anti-SYT2 N-terminal antibody" and "intravesicular domain antibody," except that the antigen is an anti-SYT2 N-terminal antibody.

[0114] The binding of a portion of an intravesicular domain antibody, such as an anti-SYT2 N-terminal antibody, to a labeling substance and / or a physiologically active substance is similar to the binding in the targeting agent or conjugate of the present invention. Therefore, the portion of an intravesicular domain antibody, such as an anti-SYT2 N-terminal antibody, to a labeling substance and / or a physiologically active substance may be linked by a non-covalent bond, directly by a covalent bond, or indirectly via a linker or the like.

[0115] When the targeting agent of the present invention contains a labeled substance and / or a bioactive substance, the labeled substance and / or the bioactive substance can be included in the targeting agent of the present invention in the form of a peptide complex in which the labeled substance and / or the bioactive substance and an intravesicular domain antibody such as an anti-SYT2 N-terminal antibody are non-covalently linked.

[0116] The site on which the labeled substance and / or bioactive substance binds to an intravesicular domain antibody, such as an anti-SYT2 N-terminal antibody, is not particularly limited, as long as it does not impair the binding of the intravesicular domain antibody to the antigen, such as binding to the SYT2 N-terminus of the anti-SYT2 N-terminal antibody. Specifically, for example, the labeled substance and / or bioactive substance can be bound to a site other than the high-frequency variable region (HVR) or to the constant region.

[0117] Furthermore, multiple labeling substances and / or physiologically active substances may be included, as long as they do not impair each other's functions. In this case, for example, multiple identical substances may be included, or one or more different substances may be included.

[0118] In the present invention, any linker that is suitably used in the art can be used as the linker. In this case, the linker configuration and chain length can be selected appropriately within a range that does not impair the function of the resulting conjugate. The linker may be configured, for example, to be cleavable after transport to the synapse. Alternatively, the linker may be configured, for example, not to be cleaved after transport to the synapse.

[0119] The linker can be any type commonly used in this field and is not particularly limited, but for example, a peptide linker consisting of 5 to 25, preferably 10 to 20, amino acid residues, such as a GS linker, can be suitably used. Alternatively, a cleavable linker such as an acid-unstable linker, a photo-unstable linker, a peptidase-sensitive linker, a dimethyl linker, or a disulfide-containing linker may be used.

[0120] Intravesical domain antibodies can bind to the intravesical domain of membrane proteins, which are antigens temporarily exposed on the cell surface by the fusion of synaptic vesicles with the cell membrane, and be delivered into the cell together with the membrane protein during endocytosis of the synaptic vesicles. The anti-SYT2 N-terminal antibody contained in the targeting agent of the present invention can bind to the intravesical domain of synaptotagmin 2, which is temporarily exposed on the cell surface by the fusion of synaptic vesicles with the cell membrane, and be delivered into the cell together with synaptotagmin 2 during endocytosis of the synaptic vesicles. Therefore, it is preferable that the targeting agent or conjugate is designed so that a labeled substance and / or a bioactive substance is delivered to the synaptic vesicles of the target synapse. The characteristics of compounds that can be delivered to synaptic vesicles are well known in the art. The particle size of the targeting agent or conjugate of the present invention can be set to, for example, 160 nm or less, 150 nm or less, 140 nm or less, 130 nm or less, 120 nm or less, 110 nm or less, 100 nm or less, or 90 nm or less, given that the diameter of endosomes in bulk endocytosis is 90 nm to 160 nm. Furthermore, given that the diameter of synaptic vesicles is 40 nm to 60 nm, the particle size of the conjugate of the present invention can be set to, for example, 60 nm or less, 55 nm or less, 50 nm or less, 45 nm or less, 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, given that the diameter of endosomes in bulk endocytosis is 90 nm to 160 nm. For example, if the targeting agent does not contain a labeling substance and / or a bioactive substance, the overall particle size when the labeling substance and / or bioactive substance binds to the targeting agent can be within the range described above. However, the particle size may be designed to be larger, for example, for the purpose of inhibiting synaptic vesicle endocytosis or for the purpose of delivering a substance to the synaptic surface or synaptic cleft. Also, for example, if the design is such that the labeling substance and / or bioactive substance is already separated by the time of delivery to the synaptic vesicle, the particle size before separation may be designed to be larger.

[0121] The conjugate or targeting agent of the present invention does not need to be configured to cross the blood-brain barrier. Typically, the conjugate or targeting agent of the present invention does not cross the blood-brain barrier and therefore does not act on the central nervous system, but can act only at synapses located in the periphery.

[0122] The targeting effect on motor neurons via synapses can be determined, for example, by administering the conjugate or targeting agent of the present invention, which contains a bioactive substance, to a target such as a vertebrate (e.g., a non-human mammal, a human, or other vertebrate) and evaluating the physiological effect of the conjugate or targeting agent on the target neurons. The evaluation of the physiological effect can be performed, for example, by comparing the degree of physiological effect between a group administered the conjugate or targeting agent of the present invention and a group that was not administered it, and / or by comparing the degree of physiological effect between a group administered the conjugate or targeting agent of the present invention and a group administered the bioactive substance alone.

[0123] The inventors have previously discovered that presynaptic formation can be induced by co-culturing nerve cells with microbeads on which LRRTM molecules (such as the extracellular domain of LRRTM2) are immobilized (WO2021 / 006075).

[0124] Therefore, whether or not the test substance has a targeting effect on motor neurons, including synapses, can also be determined by examining whether or not the test substance localizes to the presynaptic region induced by co-culturing neurons and microbeads.

[0125] "LRRTM (leucine-rich repeat transmembrane neuronal protein) family proteins" refers to proteins belonging to the LRRTM family. The LRRTM family is one of the synaptic organizer protein families on the postsynaptic side and has the activity to induce presynaptic formation. In mammals, including humans, four types of LRRTM family proteins have been reported: LRRTM1, LRRTM2, LRRTM3, and LRRTM4. Any of these LRRTM family proteins may be used in microbeads.

[0126] <Conjugate> The present invention relates to a conjugate of an antibody capable of binding to the intravesicular domain (N-terminal portion) of synaptic vesicles, such as an antibody capable of binding to the intravesicular domain (N-terminal portion) of synaptotagmin 2 (referred to as an "anti-SYT2 N-terminal antibody"), and a labeled substance and / or a bioactive substance. The conjugate of the present invention is, for example, taken up into the synaptic vesicles of motor neurons and delivered to the motor neurons.

[0127] <Visualizing agent for motor neurons or their synapses> The present invention provides a targeting agent (referred to as "the visualization agent of the present invention") which is a visualization agent for motor neurons or their synapses.

[0128] The visualization agent of the present invention is a targeting agent containing a labeling substance for use in visualizing motor nerve cells or their synapses.

[0129] "Visualizing motor neurons" refers to making all or part of motor neurons detectable. When visualizing a part of a motor neuron, the visualized part may be random or a predetermined part. For example, synapses can be visualized as a predetermined part. In that case, the visualization agent of the present invention can be used as a synapse visualization agent. "Visualizing synapses" refers to making the presynaptic and / or postsynaptic parts detectable. Therefore, the visualization agent of the present invention can use any detectable labeling substance, in addition to a labeling substance that can be directly detected by visual inspection. Similarly, the visualization agent of the present invention can visualize the axon terminal, axon, axonal papilla, cell body, dendrites, etc., of motor neurons.

[0130] The visualization agent of the present invention can be used in vivo or in vitro. Detection of the signal of the labeled substance can be performed while the motor neurons are alive or after the motor neurons have been fixed. Labeled substances suitable for detection in the state of living motor neurons are known in the art. Examples include fluorescent substances, chemical or bioluminescent substances, and other luminescent substances known in the field of in vivo imaging; sound-emitting substances such as photoacoustic effect probes; radioactive substances such as radioisotopes; and contrast agents.

[0131] The visualization agent of the present invention may be used for any purpose, for example, to visualize the number, size, or location of motor neurons or synapses (including neuromuscular junctions) or synaptic vesicles, or to visualize tissue in surgery or diagnosis.

[0132] The visualization agent of the present invention may be provided in the form of a kit together with other reagents, such as reagents necessary for detecting the labeled substance contained in the visualization agent of the present invention.

[0133] In particular, if the labeling substance is, for example, an enzyme, its substrate can be provided together with the visualization agent of the present invention.

[0134] <Composition or pharmaceutical composition> The present invention further relates to a composition ("Composition") or pharmaceutical composition ("Pharmaceutical Composition") comprising the conjugate or targeting agent of the present invention.

[0135] The composition or pharmaceutical composition of the present invention contains a targeting agent of the present invention which contains a physiologically active substance. In addition to the targeting agent, the composition or pharmaceutical composition of the present invention may optionally contain additives (for example, carriers (solid or liquid carriers, etc.), excipients, surfactants, binders, disintegrants, lubricants, solubilizers, suspending agents, coating agents, colorants, preservatives, buffers, pH adjusters, etc.). In this case, the additives can be appropriately selected according to the dosage form of the composition or pharmaceutical composition.

[0136] The composition or pharmaceutical composition of the present invention may be prepared in any dosage form, such as a solid formulation, liquid formulation, gel formulation, or aerosol formulation, but is not limited to these. When the composition or pharmaceutical composition is used as a liquid formulation, it may also be prepared as a dry product intended to be reconstituted with, for example, physiological saline solution immediately before use.

[0137] Examples of excipients include lactose, crystalline cellulose, and starch. Examples of binders include starch paste, gum arabic paste, and hydroxypropyl cellulose. Examples of disintegrants include starch, celluloses, and carbonates. Examples of lubricants include wax and talc.

[0138] When the composition or pharmaceutical composition of the present invention contains a synapse formation promoter, a synapse maintenance agent, or a muscle strengthening agent as a physiologically active substance, it can improve or prevent the decline of nerve function by promoting synapse formation. Therefore, the composition or pharmaceutical composition of the present invention can be used to improve or prevent the decline of nerve function, such as the decline of nerve function due to nerve damage, the decline of nerve function due to aging, or the decline of nerve function due to disease, or for the improvement of nerve function.

[0139] In this specification, "nerve damage" refers to damage to any part of the nerve, and includes damage caused physically from outside the body, as well as damage caused by internal factors such as cancer and tumors.

[0140] In this specification, "aging" refers to various functional declines, morphological changes, and external changes that occur in an individual organism over time, as well as the processes involved.

[0141] Frailty and sarcopenia are known conditions that arise with aging. Frailty refers to a state in which physical and mental vitality (motor function, cognitive function, etc.) declines with age, and daily living functions become impaired, and the body and mind become fragile, often due to the influence of multiple co-occurring chronic diseases. Examples of decreased physical and mental vitality include cognitive impairment, dizziness, eating disorders, swallowing disorders, visual impairment, depression, anemia, hearing loss, delirium, increased susceptibility to infection, weight loss, and decreased muscle mass. Chronic diseases include hypertension, heart disease, cerebrovascular disease, diabetes, respiratory diseases, and malignant tumors. On the other hand, sarcopenia refers to a state in which skeletal muscle mass decreases and muscle strength declines due to aging or disease. Morphological changes such as synaptic detachment and partial or complete axonal detachment from the postsynaptic terminal are observed in the neuromuscular junction of aged mice, suggesting that morphological changes in the neuromuscular junction associated with aging are involved in the decrease in skeletal muscle mass in sarcopenia.

[0142] The composition or pharmaceutical composition of the present invention can be used, in particular, to improve or prevent age-related decline in nerve function in subjects who have or are at high risk of having frailty or sarcopenia.

[0143] In the present invention, examples of diseases include neurological diseases and neuromuscular diseases.

[0144] In this specification, "neurological disease" refers to a disease caused by a disorder of the central nervous system or peripheral nervous system, and for example, one or more diseases selected from the group consisting of: Alzheimer's disease, Parkinson's disease, Lewy body dementia, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration, Huntington's disease, dystonia, prion disease, acanthocyte chorea, adrenoleukodystrophy, multiple system atrophy, spinocerebellar degeneration, amyotrophic lateral sclerosis, primary lateral sclerosis, spinal and bulbar muscular atrophy, spinal muscular atrophy, spastic paraplegia, syringomyelia, Charcot-Marie-Tooth disease, frontotemporal dementia, epilepsy, schizophrenia, autism, autism spectrum disorder, etc.

[0145] In this specification, “neuromuscular disease” refers to a disease resulting from a disorder of motor nerves, neuromuscular junctions, or muscle cells, and includes, for example, one or more diseases selected from the group consisting of: muscular dystrophy, myopathy, congenital myasthenic syndrome, hereditary periodic paralysis, myasthenia gravis, Lambert-Eaton syndrome, etc.

[0146] Amyotrophic lateral sclerosis (ALS) is a disease in which primary and secondary motor neurons selectively and progressively degenerate and disappear, and it is known that in the early stages of the disease, motor neurons detach from skeletal muscle at the neuromuscular junction. Therefore, the composition or pharmaceutical composition of the present invention, which contains a synapse formation promoter or synapse maintenance agent that can promote the formation of synapses between skeletal muscle and motor neurons or suppress their regression, can be used in particular to improve or prevent the decline in nerve function caused by amyotrophic lateral sclerosis.

[0147] For example, the pharmaceutical composition of the present invention may be a pharmaceutical composition for the treatment of amyotrophic lateral sclerosis, a pharmaceutical composition for the treatment of spinal muscular atrophy, etc., containing the conjugate or targeting agent of the present invention.

[0148] The composition or pharmaceutical composition of the present invention, which contains a muscle-enhancing agent, can improve or prevent muscle weakness by enhancing muscle strength. Therefore, the composition or pharmaceutical composition of the present invention, which contains a muscle-enhancing agent, can be used to improve or prevent muscle weakness, such as muscle weakness after trauma or surgery, muscle weakness due to aging, or muscle weakness due to disease, or to improve muscle function.

[0149] In this specification, "trauma" refers to damage to tissue or organs caused by external factors, including, for example, wounds, fractures, sprains, internal organ ruptures, burns, frostbite, etc.

[0150] The composition or pharmaceutical composition of the present invention, containing a functional modifier, can improve or prevent the decrease or increase of nerve function by modifying the function of motor nerve cells. Therefore, the composition or pharmaceutical composition of the present invention, containing a functional modifier, can also be used to improve or prevent nerve hyperactivity, for example, nerve hyperactivity due to disease or condition, or to improve or prevent muscle tension, for example, tremors due to aging, muscle tension due to trauma or disease.

[0151] Specifically, in addition to the various diseases and conditions mentioned above, it can be used to improve or prevent abnormal involuntary movements (dyskinesia) (e.g., abnormal head movements, tremors, (painful) spasms, fasciculations, etc.), abnormalities in gait and mobility (e.g., ataxic gait, difficulty walking, etc.), other coordination disorders (e.g., ataxia, etc.), and other conditions relating to the nervous and musculoskeletal systems (e.g., tetany, abnormal reflexes, postural abnormalities, spasticity, hypertonia, muscle tone, hypertonia, hyperextended deep tendon reflexes, dysphagia, etc.).

[0152] In this specification, "disease" refers to a pathological condition that can be classified by identifiable symptoms or causes in the subject individual, and includes diseases and disorders. In the present invention, "condition" refers to a pathological condition in the subject individual that includes identifiable symptoms but does not fall under the category of disease.

[0153] The composition or pharmaceutical composition of the present invention may contain a plurality of the targeting agents of the present invention, and may further contain other active ingredients. The other active ingredients are not particularly limited as long as they do not impair the function of the targeting agents contained in the composition or pharmaceutical composition. When multiple types of targeting agents are included, the membrane proteins to which each targeting agent can bind may be the same or different. Furthermore, for example, even if the same membrane protein is used, the composition may include a targeting agent containing an antibody that can bind to different sites. The labeling substances and / or physiologically active substances contained in these targeting agents may be different from each other or the same.

[0154] <Method for targeting motor neurons or synapses> The present invention relates to a method for targeting motor neurons or synapses. According to the present invention, by bringing an antibody capable of binding to the intravesicular domain of a membrane protein present in synaptic vesicles (referred to as an "intravesicular domain antibody"), such as an antibody that binds to the intravesicular domain of synaptotagmin 2 (anti-SYT2 N-terminal antibody), into contact with a motor neuron, the antibody can be taken up into the motor neuron (particularly within the synaptic vesicles of the motor neuron), and targeted to the motor neuron (for example, the axon terminal, axon, axonal papilla, cell body, dendrites, etc. of the motor neuron). In this way, intravesicular domain antibodies, including antibodies that bind to the intravesicular domain of synaptotagmin 2, are targeted to motor neurons or synapses. The present invention provides a conjugate to which a desired substance (e.g., a labeled substance and / or a bioactive substance) can be directly or indirectly linked via a linker to the antibody. The conjugate can then be brought into contact with a motor neuron, or the antibody and the desired substance capable of binding to the antibody can be brought into contact with the motor neuron separately, thereby delivering the desired substance into the motor neuron (e.g., within the synaptic vesicles of the motor neuron). Therefore, the present invention provides a method for targeting motor neurons or synapses, which includes bringing an antibody (which may be linked to the desired substance) into contact with a cell such as a motor neuron.

[0155] <Method for targeting labeled substances and / or physiologically active substances> The present invention relates to a method for targeting a labeled substance and / or a physiologically active substance, comprising the steps of contacting a motor neuron with the conjugate or targeting agent of the present invention, and delivering the targeting agent to the synapse of the motor neuron.

[0156] The target of contact in the present invention is not particularly limited as long as it includes motor neurons. Contact may be made, for example, with individual motor neurons or with tissue containing cells other than motor neurons. For example, since the targeting agent of the present invention primarily targets the presynaptic portion of motor neurons at the neuromuscular junction, the target may also be tissue further containing skeletal muscle cells to which motor neurons project.

[0157] The motor neurons used in this method may include motor neurons from vertebrates (non-human mammals, humans, and other vertebrates). Preferably, the motor neurons used in this method include human motor neurons. The present invention will be described below using human motor neurons as an example, but this method is not limited to human motor neurons.

[0158] Human motor neurons can be used without restriction, regardless of their origin. For example, but are not limited to, primary cultures of cells isolated from humans, cells isolated from humans and established as cell lines, and human motor neurons differentiated from human-derived pluripotent stem cells. The pluripotent stem cells from which human motor neurons are derived are preferably human-derived pluripotent stem cells.

[0159] The term "pluripotent stem cells" as used in this invention refers to cells that have the ability to self-renew, can be cultured in vitro, and possess the multipotency to differentiate into cells that constitute an individual. Specifically, examples include embryonic stem cells (ES cells), pluripotent stem cells derived from fetal primordial germ cells (GS cells), and induced pluripotent stem cells (iPS cells) derived from somatic cells, but human-derived iPS cells or ES cells are preferably used in this method.

[0160] ES cells are often obtained from fertilized eggs, but they can also be obtained from other sources, such as adipose tissue, placenta, and testicular cells, and all types of ES cells are subject to the present invention. Methods for producing ES cells from sources other than fertilized eggs have been reported (e.g., WO2003 / 046141), and these reports can be used by referring to them as appropriate.

[0161] 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 they exhibit characteristics almost equivalent to those of ES cells (e.g., pluripotency and proliferative ability based on self-renewal). Examples of genes included in the reprogramming factors include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-MYC, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, Glis1, or combinations thereof. Many reports have been made on methods for producing iPS cells, and these reports can be referred to and modified as appropriate. The iPS cells that can be used in this method are preferably human-derived iPS cells, for example, iPS cells derived from human fibroblasts.

[0162] Many methods have been reported for differentiating human iPS cells into peripheral nerve cells (e.g., Chambers, Stuart M., et al., Nature biotechnology 27.3 (2009): 275.), and these reports can be referenced and modified as appropriate. Alternatively, various types of nerve cells differentiated from human iPS cells are commercially available and can be purchased from companies such as FUJIFILM Cellular Dynamics, Inc. or ReproCELL, Inc.

[0163] In one embodiment, this method is an in vitro method. In another embodiment, this method is an ex vivo method. In yet another embodiment, this method is an in vivo method. When this method is an in vivo method, the subject may be a mammal other than a human.

[0164] If the present method is an in vitro method, the method may further include a step of inducing synapse formation. In this specification, “inducing synapse formation” means causing the formation of a presynaptic terminal in the axon of a nerve cell and / or causing the formation of a postsynaptic terminal in the dendrites of another nerve cell or in cells of skeletal muscle, organs, etc. Induction of synapse formation can also be carried out by co-culturing cells that intend to form a presynaptic terminal with cells that intend to form a postsynaptic terminal. Alternatively, induction of synapse formation can be carried out by other methods, for example, by co-culturing motor neurons with beads coated with the extracellular domain of LRRTM2. The step of inducing synapse formation may be carried out simultaneously with or before the step of contacting motor neurons with the targeting agent of the present invention.

[0165] When this method is an in vitro method, the step of contacting motor neurons with the targeting agent of the present invention is performed by contacting a sample containing motor neurons with the targeting agent of the present invention. The method of contact is not particularly limited as long as the motor neurons in the sample and the targeting agent can come into contact with each other. For example, the targeting agent can be applied by directly spraying, atomizing, dropping, or coating the sample, by immersing the sample in the targeting agent, or by a combination thereof. Furthermore, if the sample is in another carrier (e.g., culture medium), the targeting agent may be applied by spraying, atomizing, dropping, or coating the carrier.

[0166] The applicable dose is not particularly limited and can be set appropriately considering the number of motor neurons and other conditions. For example, concentrations of IgG antibody of 0.01 μg / mL or higher, 0.1 μg / mL or higher, 0.2 μg / mL or higher, 0.5 μg / mL or higher, 0.7 μg / mL or higher, 0.9 μg / mL or higher, 1 μg / mL or higher, 2 μg / mL or higher, 5 μg / mL or higher, 7 μg / mL or higher, 9 μg / mL or higher, or 10 μg / mL or higher can be applied.

[0167] When this method is an in vivo method, the step of contacting the test sample with the targeting agent of the present invention is performed by administering the targeting agent of the present invention.

[0168] The method of administration is not particularly limited, but examples include local administration, enteral administration, and parenteral administration. Specifically, these include administration on the skin, by inhalation, enema, eye drops, ear drops, nasal administration, vaginal administration, tube feeding, intravenous administration, intraarterial administration, intramuscular administration, intracardiac administration, subcutaneous administration, intraosseous administration, intradermal administration, subarachnoid (cavity) administration, intraperitoneal administration, intravesical administration, transdermal administration, transmucosal administration, epidural administration, intravitreous administration, etc.

[0169] The dosage is not particularly limited and can be set as appropriate, taking into account the target animal species and other conditions. For example, when administering IgG antibodies to mice, 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 kg of body weight can be applied.

[0170] The targeting agent used in this process does not need to be of a single type. For example, multiple types of targeting agents can be used together or separately. Specifically, for example, a targeting agent containing a conjugate and a targeting agent that does not contain a labeling substance and / or a bioactive substance can be used. Furthermore, the labeling substance and / or bioactive substance used does not need to be of a single type; for example, multiple types of labeling substances and / or bioactive substances can be used together or separately. Specifically, for example, a combination of a labeling substance and a bioactive substance may be used.

[0171] The step of contacting motor nerve cells with the targeting agent of the present invention can be performed multiple times. When this step is performed multiple times, the type of targeting agent and cells used, as well as the application method and administration method, may be the same or different each time.

[0172] When the conjugate or targeting agent of the present invention is brought into contact with motor neurons, the targeting agent is taken up by the motor neurons via synaptic vesicles. When the targeting agent of the present invention is brought into contact with motor neurons, it can activate or promote the activity of the motor neurons. By activating or promoting the activity of motor neurons, the efficiency of taking up the conjugate or targeting agent of the present invention into motor neurons can be improved. Normally, once the conjugate or targeting agent of the present invention is taken up by synaptic vesicles, it is delivered to the cell body via retrograde transport through the axon.

[0173] The method for activating motor neurons is not particularly limited, but examples include methods for spontaneously activating motor neurons for a sufficient period of time, and methods for activating motor neurons or promoting synaptic vesicle endocytosis through artificial stimulation.

[0174] Methods for inducing spontaneous activity include, for example, placing motor neurons in an environment where they can be active for a sufficient period of time. Environments in which motor neurons can be active are well known in this art.

[0175] In the method of spontaneously activating motor neurons, the time for activating motor neurons (for example, the contact time with the conjugate or targeting agent of the present invention) is not particularly limited, but for example, if the method is an in vitro method, it can be 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. Also, if the method is an in vivo method, for example, it can be 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.

[0176] Methods for activating or promoting the activity of motor neurons through artificial stimulation include, for example, placing motor neurons in an environment where they can be actively engaged for a sufficient period of time.

[0177] If this method is an in vitro method, the method for activating or promoting the activity of motor neurons can involve applying chemical and / or physical stimuli to the motor neurons. Stimuli for activating motor neurons are well known in the art. Examples of compounds used for chemical stimuli include potassium ion channel inhibitors such as amiodarone, tetraethylammonium, 4-aminopyridine, barium, and dendrotoxin, sodium channel agonists such as batrachotoxin, calcium channel agonists such as Bay K8644, high concentrations of potassium ions or neurotransmitters, or combinations thereof. Examples of physical stimuli include temperature changes.

[0178] When performing chemical stimulation, there are no particular limitations on the amount of compound added. For example, it can be added at concentrations of 1 μM or higher, 10 μM or higher, 50 μM or higher, or 100 μM or higher.

[0179] The duration of stimulation is not particularly limited and can be set as appropriate, taking into account conditions such as the type and intensity of the stimulation. For example, stimulation can be applied 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.

[0180] If this method is an in vivo method, the method for activating or promoting the activity of motor neurons can be carried out by methods that make the subject actively move (for example, by making the subject move or by methods that stimulate brain activity), or by methods that promote the activity of motor neurons using chemical substances, etc. Examples of compounds that promote the activity of motor neurons include the compounds used in the above-mentioned chemical stimulation, and these can be administered to the subject. The compounds used in the above-mentioned chemical stimulation can be administered at pharmaceutically acceptable concentrations or in a manner. The above-mentioned compounds can be administered to the subject in such a way that the compound stimulation does not exert a biological toxicity, but if a biological toxicity occurs, it is not necessary to administer the above-mentioned compounds to the subject. When the activity of motor neurons is promoted, a similar effect can usually be expected in a shorter time compared to methods that induce spontaneous firing.

[0181] When using a targeting agent that does not contain a labeling substance and / or a bioactive substance, the labeling substance and / or bioactive substance and the targeting agent can be brought into contact with motor neurons together or separately. When the labeling substance and / or bioactive substance and the targeting agent are brought into contact separately, the timing is not particularly limited as long as the labeling substance and / or bioactive substance can bind to the targeting agent. For example, contact with the labeling substance and / or bioactive substance can be performed before or after contact with the targeting agent.

[0182] Each contact method can be selected in accordance with the contact methods described above. For example, the same method or different methods can be used for each contact.

[0183] This method may further include a step to confirm the success or failure of targeting, if necessary. If the targeting agent used in this method contains a physiologically active substance, for example, as described above in the section on targeting agents, targeting can be determined to have been successful if a physiological effect is observed. Furthermore, if the targeting agent used in this method contains a labeling substance, targeting can be determined to have been successful if a signal is detected, in accordance with the step of detecting the signal of the labeling substance in the visualization method described later.

[0184] When the pharmaceutical composition of the present invention is used as the targeting agent in this method, this method can be used as a method for preventing or treating a condition or disease.

[0185] In this case, the present invention relates to a method for preventing or treating a condition or disease, comprising the steps of contacting a motor neuron with a targeting agent containing an antibody and a bioactive substance capable of binding to the intravesicular domain of a membrane protein present in the synaptic vesicles of a motor neuron, and delivering the targeting agent to the synapse of the motor neuron. According to this method, various conditions or diseases exemplified with respect to the pharmaceutical composition can be prevented or treated. The contacting step of this method preferably includes administering the targeting agent and / or the pharmaceutical composition to the target.

[0186] Therefore, for example, the method of the present invention is a method for improving or preventing 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 for improving nerve function. In one embodiment, the condition or disease is a condition or disease that exhibits a decline in nerve function. In one embodiment, the condition or disease is a neurological disease and a neuromuscular disease. In one embodiment, the targeting agent comprises a conjugate of the antibody and the physiologically active substance.

[0187] In this case, the process may further include steps to allow the physiological effects to be fully exerted in the subject. For example, if the physiologically active substance used is a substance that can exert its effects on its own, the effect can be achieved by placing the subject in a well-nourished environment for a sufficient amount of time for the physiological effects to be exerted. Also, for example, if other substances are necessary for the physiologically active substance to exert its effects, those substances can be administered additionally.

[0188] The duration of this process can be appropriately determined depending on the condition of the subject, the type of physiologically active substance, the dosage, etc. For example, it may be determined based on the time it takes for physiological effects to be exerted when a physiologically active substance is administered in general, or the physiological effect may be confirmed once or multiple times and continued until the physiological effect is fully exerted.

[0189] <Method for visualizing motor neurons or their synapses> The present invention relates to a method for visualizing motor neurons or synapses, comprising the steps of: contacting a motor neuron with a visualization agent of the present invention; delivering the visualization agent to a motor neuron synapse; and detecting the signal of the labeling substance.

[0190] In this method, the motor neurons used for contact are those described in the above-mentioned method for targeting the labeled substance and / or bioactive substance.

[0191] In one embodiment, the method is an in vitro method. In another embodiment, the method is an ex vivo method. In yet another embodiment, the method is an in vivo method. When the method is an in vivo method, the subject may be a human or a mammal other than a human.

[0192] In this method, the steps of contacting the visualization agent of the present invention with motor neurons and delivering the visualization agent to the synapses of motor neurons are the same as the steps of contacting the targeting agent of the present invention with motor neurons and delivering the targeting agent to the synapses of motor neurons as described above in the method for targeting labeled substances and / or physiologically active substances, except that the visualization agent is used as the targeting agent.

[0193] This method may further include a step of generating a signal from a labeled substance, if necessary. The method of generating the signal is not particularly limited. The method of generating the signal and whether or not it is necessary can be determined based on the type of labeled substance used, etc.

[0194] For example, if the labeling substance is a fluorescent molecule or a radiolabeling substance, a signal can be generated at the target by waiting for a sufficient amount of time for the visualization agent to be delivered to the target motor neuron synapse, that is, for a sufficient amount of time for the visualization agent to reach the target motor neuron synapse and for sufficient endocytosis of synaptic vesicles to occur at that synapse. This time can be appropriately selected in accordance with the time required for the process of delivering the visualization agent to the motor neuron synapse.

[0195] For example, if the labeling substance is a chemiluminescent substance, this can be done by waiting long enough for the visualization agent to be delivered to the target motor neuron cell synapse, in addition to adding a substance such as its substrate used to generate the signal.

[0196] This process can be performed simultaneously with or before the signal detection process described later.

[0197] This method further includes a 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.

[0198] If the labeling substance is a fluorescent substance, for example, it can be detected by irradiating motor neurons with excitation light containing the excitation wavelength of the labeling substance and using a detector capable of detecting the fluorescence wavelength of the labeling substance. If the labeling substance is a chemiluminescent substance, for example, it can be detected using a detector capable of detecting the emission wavelength of the labeling substance. If the labeling substance is a radioactive labeling substance, it can be detected using a detector capable of detecting the radiation emitted by the labeling substance.

[0199] The detection of a labeled substance's signal includes detecting the presence, location, or quantity of motor neurons (e.g., synapses, cell bodies, etc.) in a sample containing motor neurons.

[0200] The method of the present invention may further include, in addition to the step of detecting the signal of a labeled substance in a sample, a step of comparing the signal of the labeled substance detected in the sample with the signal in a standard sample containing the labeled substance, or a pre-established reference value, to determine its 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 particular condition or disease exists. Specifically, examples include those obtained from healthy individuals, those obtained from the same individual as the sample at different collection times, or those obtained from individuals known to have a particular condition or disease. The standard sample may be a biological sample derived from the same species, individual, tissue, or cell as the sample, or a biological sample derived from a different species, individual, tissue, or cell. Furthermore, the reference value is not particularly limited as long as it is a value that serves as a standard for determining whether or not the desired condition exists. The reference value can be set, for example, based on the intensity or number of signals generally detected in the standard sample.

[0201] In any case, the method of comparison is not particularly limited. For example, it can be done visually, by comparing the magnitude of the values, or by using statistical methods.

[0202] <Other Inventions> The present invention provides a method for administering a substance to a target. 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 target cells, such as motor neurons. If the substance is a bioactive substance, it can be delivered to cells such as motor neurons. If the substance is a labeling substance, it can be used to observe the delivery site of the labeling substance (e.g., motor neurons or their synapses). The present invention also provides a conjugate of the antibody and the substance for use in this method, or a composition containing the conjugate. In this case, the present invention relates to a targeting composition for motor neurons or their synapses, comprising an antibody capable of binding to the intravesicular domain of a membrane protein present in synaptic vesicles of motor neurons, or the above antibody and a conjugate of a labeling substance and / or a bioactive substance.

[0203] The present invention provides a method for visualizing target motor neurons, comprising administering to the target an effective amount of a conjugate of an intravesicular domain antibody, such as an anti-SYT2 N-terminal antibody, and a labeling substance. 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.

[0204] The present invention provides a method for delivering a bioactive substance to target motor neurons, comprising administering to the target an effective amount of a conjugate of an intravesicular domain antibody, such as an anti-SYT2 N-terminal antibody, and the bioactive substance. The present invention also provides a conjugate of the antibody and the bioactive substance, or a composition containing the conjugate, for use in this method.

[0205] The present invention provides the intravesicular domain antibody or a conjugate of the intravesicular domain antibody and the labeling substance and / or bioactive substance for use in any of the above methods. For example, the present invention relates to the intravesicular domain antibody or a conjugate of the intravesicular domain antibody and the bioactive substance for use in a method for preventing or treating a condition or disease. Also, for example, the present invention relates to the intravesicular domain antibody or a conjugate of the intravesicular domain antibody and the labeling substance and / or bioactive substance for use in a method for targeting a labeling substance and / or bioactive substance to motor neurons or their synapses.

[0206] The present invention provides the above-mentioned antibody or a conjugate of the above-mentioned antibody and the above-mentioned substance for use in the manufacture of a pharmaceutical product for use in any of the above-mentioned methods.

[0207] Furthermore, the present invention provides the use of the intravesicular domain antibody or a conjugate of the intravesicular domain antibody and a bioactive substance in the manufacture of a pharmaceutical product containing the antibody and the bioactive substance. [Examples]

[0208] The present invention will be described in more detail below using examples. However, the technical scope of the present invention is not limited to these examples.

[0209] <Example 1: Delivery of synaptotagmin 2 antibody to human motor neuron synapses> We investigated whether synaptotagmin 2 antibody can be delivered to human motor neuron synapses, and under what conditions, using microbeads coated with the extracellular domain of LRRTM2.

[0210] 1. Cell culture Human iPS-induced motor neurons (40HU-005-2M; ixcells biotechnologies) were thawed using the Dead Cell Removal Kit (Veritas) according to the kit's protocol. The thawed cells were placed in 2 × 10⁶ well plates (V-bottom).4 Neurospheres were created by seeding cells at a cell / well density and culturing them for one week in Motor Neuron Maintenance Medium (ixcells biotechnologies). The created neurospheres were selected based on size and roundness, and those meeting these criteria were used in the following experiments.

[0211] 96-well EZVIEW® culture plates LB (AGC Techno Glass Co., Ltd.) were coated with poly-D-lysine and Geltrex® Matrix (Thermo Fisher Scientific). Selected neurospheres were then seeded onto the plates and cultured for 20 days. Initially, the motor neuron culture medium described above was used, and the medium was changed with the same medium on the second day of culture. Subsequently, neuronal medium (Neurobasal plus medium (B27 plus supplement (Thermo Fisher Scientific), supplemented with 20 ng / mL BDNF, 20 ng / mL GDNF, and penicillin-streptomycin)) was used, and the medium was changed three times a week. Each medium change was performed with 50 μL / well.

[0212] 2. Preparation of LRRTM2 beads Microbeads coated with the extracellular domain of LRRTM2 (active beads) were prepared as disclosed in WO2021 / 006075.

[0213] Specifically, streptavidin-coated microspheres (Bangs Laboratories, Inc; made of polystyrene, average diameter 9.94 μm) were washed twice with a washing buffer (phosphate-buffered saline (PBS), 0.01% bovine serum albumin (BSA), 0.05% TritonX-100), and then reacted with biotinylated anti-human IgG (Fc-specific) antibody (Sigma-Aldrich; mouse monoclonal) in a binding buffer (PBS, 0.01% BSA) to immobilize the streptavidin-coated microspheres with the biotinylated anti-human IgG (Fc-specific) antibody. The resulting beads were washed three times with the washing buffer (anti-human IgGFc antibody beads).

[0214] Next, anti-human IgGFc antibody beads were suspended in 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 Inc.) was added to the suspension, immobilizing LRRTM2-Fc onto the anti-human IgGFc antibody beads. The resulting beads were washed with washing buffer and suspended in binding buffer (LRRTM2 bead suspension).

[0215] 3. Presynaptic induction (Figure 1) LRRTM2 beads were seeded at a concentration of 0.1 μg / well in plates that had been cultured for 20 days, and the cells were incubated at 37°C for 48 hours to induce presynaptic formation.

[0216] 4. Preparation of antibody solution As antibodies, we used either a rabbit anti-SYT2 N-terminal antibody (Polyclonal rabbit purified antibody SYT2 lumenal domain; catalog number 105 223; Synaptic Systems) capable of binding to the peptide having the amino acid sequence of SEQ ID NO: 5, or, as a control, a normal rabbit IgG antibody (Normal Rabbit IgG; catalog number AB-105-C; R&D Systems).

[0217] After warming the neuronal culture medium at 37°C for 30 minutes, antibody was added to the medium to a final concentration of 1 μg / mL or 10 μg / mL and mixed. This crude antibody solution was centrifuged at 200 g for 3 minutes at room temperature, and the supernatant was collected as the antibody solution.

[0218] 5. Introduction of antibodies Antibodies were introduced into plates containing presynaptic terminals by changing the culture medium with 100 μL of antibody solution.

[0219] 6. Spontaneous activity of motor neurons After introducing antibodies, spontaneous activity of motor neurons was promoted by culturing them at 37°C for 24 hours.

[0220] 7. Cell fixation After culturing, the cells were washed with neuronal culture medium and then fixed with 2% paraformaldehyde (PFA) solution.

[0221] 8. Immunocytochemical staining The added antibody was used as the primary antibody for staining by immunocytochemistry. After fixing the cells, the cell membrane was permeabilized with a surfactant and blocked, followed by a primary antibody reaction using mouse anti-βIII tubulin (Tuj1) antibody (catalog number 801202; Biolegend) as an additional primary antibody. Subsequently, secondary antibody reactions were performed against each primary antibody, and fluorescence images were obtained. Blocking was performed using a blocking buffer (PBS + 2% normal goat serum + 1% BSA + 1% fetal bovine serum + 0.02% TritonX-100).

[0222] The secondary antibodies used are as follows: Alexa 488-labeled anti-rabbit antibody (catalog number A32731; Thermo Fisher Scientific); Alexa 555-labeled anti-mouse antibody (catalog number A32727; Thermo Fisher Scientific).

[0223] Fluorescence images were acquired using an inverted live-cell (DMi8) fluorescence microscope (Leica) equipped with LAS X software (Leica), with the following excitation wavelength, detection wavelength, exposure time, and detection threshold. A gamma correction value of 1 was used for all settings.

[0224] 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.

[0225] The results are shown in Figures 2-1 and 2-3. Figures 2-1 and 2-3 show immunocytochemical staining images at antibody concentrations of 1 μg / mL and 10 μg / mL, respectively.

[0226] In the control group using normal rabbit IgG antibody, no signal was observed at any concentration (Figures 2-1A and 3A). On the other hand, when anti-SYT2 N-terminal antibody was used, a strong signal was observed on the LRRTM2 beads at all concentration conditions (Figures 2-1B and 3B). Only the presynaptic portion of motor neurons was present on the LRRTM2 beads, with no postsynaptic portion. This indicates that the antibody is delivered to the surface of the LRRTM2 beads by targeting the SYT2 N-terminus. Furthermore, since sufficient time had elapsed for synaptic vesicle endocytosis, it is thought that the introduced anti-SYT2 N-terminal antibody was taken up by the motor neuron synaptic vesicle.

[0227] Furthermore, Figure 2-2 shows a magnified view of LRRTM2 beads in an experiment in which anti-SYT2 N-terminal antibody was administered at a concentration of 1 μg / mL. In the LRRTM2 beads where nerve axons are not densely packed, indicated by Tuj1 (Figure 2-2A), nerve axon terminals (the strong signal points for Tuj1 in the figure) are not observed on the surface of the LRRTM2 beads. From this, it is considered that synapse formation does not occur on the surface of these LRRTM2 beads. On the other hand, in the LRRTM2 beads where nerve axons are densely packed (Figure 2-2B), nerve axon terminals are observed on the surface of the LRRTM2 beads. From this, it is considered that synapse formation occurs on the surface of these LRRTM2 beads. No signal of anti-SYT2 N-terminal antibody was detected on the surface of the LRRTM2 beads in Figure 2-2A, but the same signal was observed on the surface of the LRRTM2 beads in Figure 2-2B. This indicates that by targeting the SYT2 N-terminus, the antibody is delivered to the surface of the LRRTM2 beads only when the presynaptic terminal of a motor neuron is present.

[0228] Furthermore, the signal intensity was higher at an antibody concentration of 10 μg / mL (Figure 3B) compared to 1 μg / mL (Figure 2-1B), indicating that the amount of antibody in the presynaptic region increases in a concentration-dependent manner with the added antibody.

[0229] These results indicate that antibodies against the intravesicular domain of synaptotagmin 2 are delivered to the presynaptic terminal of motor neurons, and that the amount delivered depends on the antibody concentration.

[0230] <Example 2: Delivery of synaptotagmin 2 antibody to human motor neuron synapses by cell stimulation> We investigated whether synaptotagmin 2 antibodies are delivered to human motor neuron synapses upon stimulation of nerve cells.

[0231] Cell culture, preparation of LRRTM2 beads, and induction of the presynaptic terminal were performed in the same manner as in Example 1.

[0232] An antibody solution containing 4-aminopyridine was used to stimulate nerve cells. The antibody solution was prepared by the following method.

[0233] After warming the neuronal culture medium at 37°C for 30 minutes, 4-aminopyridine (Sigma Aldrich) was added to the medium to a final concentration of 100 μM and mixed. Furthermore, the antibody was added to a final concentration of 2 μg / mL and mixed. This crude antibody solution was centrifuged at 200 g for 3 minutes at room temperature, and the supernatant was collected as the antibody solution.

[0234] Antibodies and 4-aminopyridine were introduced into plates containing synapses by changing the culture medium with 100 μL of antibody solution.

[0235] Motor neurons were stimulated by introducing antibodies and 4-aminopyridine, followed by incubation at room temperature for 10 or 30 minutes.

[0236] Subsequent cell fixation and immunocytochemical staining were performed in the same manner as in Example 1. The experiment concerning temperature conditions was conducted in the same manner as described above, except for the temperature.

[0237] For the 37°C temperature condition, motor neurons were stimulated by culturing them at 37°C for 30 minutes after introducing the antibody and 4-aminopyridine.

[0238] For the 4°C temperature condition, motor neurons were stimulated by culturing them at 4°C for 30 minutes after introducing the antibody and 4-aminopyridine.

[0239] For some motor neurons, the motor neurons were stimulated by introducing antibodies and 4-aminopyridine, culturing them at 4°C for 30 minutes, and then raising the temperature to 37°C and culturing them for another 30 minutes.

[0240] The results are shown in Figures 4 and 5. Figures 4 and 5 show immunocytochemical staining images for reaction times of 10 minutes and 30 minutes, respectively.

[0241] In the control group using normal rabbit IgG antibody, no signal was observed under any time conditions (Figures 4A and 5A). On the other hand, when anti-SYT2 N-terminal antibody was used, a signal was observed in the LRRTM2 beads under all time conditions (Figures 4B and 5B). This indicates that anti-SYT2 N-terminal antibody is delivered to the presynaptic terminal not only by the spontaneous activity of motor neurons but also by chemical stimulation of motor neurons.

[0242] Furthermore, the signal intensity was slightly higher when the reaction time was 30 minutes (Figure 5B) compared to 10 minutes (Figure 4B), indicating that the amount of antibody in the presynaptic terminal increases with longer stimulation times. Additionally, when a similar experiment was performed under low-temperature conditions (4°C) where cell activity is significantly reduced, the signal of the anti-SYT2 N-terminal antibody in the LRRTM2 beads was significantly lower compared to the experiment at 37°C. This decrease in signal was recovered by returning the temperature from 4°C to 37°C.

[0243] These results indicate that antibodies against the intravesicular domain of synaptotagmin 2 are delivered to the presynaptic terminal of motor neurons in a neuronal activity-dependent manner, and that the amount delivered depends on the reaction time.

[0244] <Example 3: Delivery of synaptotagmin 2 antibody to mouse motor neuron synapses by intravenous injection> We investigated whether synaptotagmin 2 antibody, introduced into the body by intravenous injection, is delivered to the synapses of motor neurons.

[0245] The antibody used for introduction was the same as that used in Example 1. The antibody solution used was prepared by adding antibody to PBS and mixing it until the final concentration was 1 mg / mL.

[0246] The antibody solution was administered to wild-type mice by tail vein injection at a dose of 5 mg / kg. Mice were sacrificed 12, 24, or 72 hours after administration, and their calf muscles were harvested and fixed.

[0247] The fixed gastrocnemius muscle was frozen with tissue embedding medium, and 10 μm thick sections were prepared from this frozen tissue block using a cryostad.

[0248] The administered antibody was used as the primary antibody for immunohistochemical staining. After blocking the prepared tissue sections, primary antibody reactions were performed using α-bungarotoxin antibody (α-BgtX antibody Alexa Fluor 594 conjugate; catalog number B13423; Thermo Fisher Scientific) and synapsin 1 antibody (SYN1 antibody; catalog number 106 308; Synaptic Systems) as additional primary antibodies. Subsequently, secondary antibody reactions were performed against each primary antibody, and fluorescence images were obtained. Blocking was performed using blocking buffer (PBS + 2% normal goat serum + 1% BSA + 1% fetal bovine serum + 0.02% TritonX-100).

[0249] The following antibodies were used as secondary antibodies: Alexa 488-labeled anti-rabbit antibody (catalog number A32731; Thermo Fisher Scientific); Alexa 647-labeled anti-guinea pig antibody (catalog number A21450; Thermo Fisher Scientific).

[0250] Fluorescence images were acquired in essentially the same manner as in Example 1. For detecting the Alexa 647 signal, a maximum excitation wavelength of 650 nm, a maximum detection wavelength of 665 nm, and an actual detection wavelength of 705 nm were used. For detecting the Alexa 594 signal, a maximum excitation wavelength of 590 nm, a maximum detection wavelength of 617 nm, and an actual detection wavelength of 595 nm were used. The following exposure times and detection thresholds were used for detecting each signal. A gamma correction value of 1 was used for all cases.

[0251] Exposure time and detection threshold: Alexa 488 exposure time: 150 ms; detection threshold: 130 - 1500; Alexa 594 exposure time: 100 ms; detection threshold: 100 - 2500; Alexa 647 exposure time: 100 ms; detection threshold: 130 - 1000.

[0252] The results are shown in Figures 6 and 7. Figures 6 and 7 show immunohistochemical staining images 12 hours and 72 hours after administration, respectively. Here, α-BgtX is a marker for acetylcholine receptors on the muscle cell membrane, and SYN1 is a marker for the presynaptic terminal.

[0253] In the control using normal rabbit IgG antibody as the antibody, no signal of normal rabbit IgG antibody was observed at the neuromuscular junction where signals of α-BgtX and SYN1 were observed under any time conditions (Figures 6A and 7A).

[0254] On the other hand, when the anti-SYT2 N-terminal antibody was used as the antibody, signals of the anti-SYT2 N-terminal antibody were observed at the neuromuscular junction under any time conditions (Figures 6B and 7B).

[0255] Synaptotagmin 2 is known to be expressed in the presynaptic terminal of motor neurons. From this, it was found that the anti-SYT2 N-terminal antibody administered by intravenous injection was delivered to the presynaptic terminal of motor neurons in vivo. Also, since sufficient time has passed for endocytosis of synaptic vesicles, it is considered that the administered anti-SYT2 N-terminal antibody has been taken up into the synaptic vesicles of motor neurons.

[0256] In addition, since the signal intensity was slightly higher in the case of 72 hours (Figure 7B) compared to the case of 12 hours after antibody administration (Figure 6B), it was found that the amount of antibody in the presynaptic terminal increases as the exposure time to the antibody becomes longer.

[0257] Furthermore, even 72 hours after antibody administration, no signals from the anti-SYT2 N-terminal antibody were detected in the mice or in the liver, which is the projection target of the autonomic nervous system, and no abnormalities were observed.

[0258] The anti-SYT2 N-terminal antibody used is a complete IgG antibody and therefore does not cross the blood-brain barrier. Furthermore, synaptotagmin 2 is known not to be expressed in sensory nerve cells. In addition, since no signal was observed in the liver, it was found that the antibody against the intravesicular domain of synaptotagmin 2 is delivered to the presynaptic terminal of motor nerve cells by intravenous injection, and that antibody administration does not cause significant side effects, and that the amount of antibody depends on the exposure time to the antibody.

[0259] <Example 4: Delivery of synaptotagmin 2 antibody to mouse motor neuron synapses by intraperitoneal injection> We investigated whether synaptotagmin 2 antibody, introduced into the body by intraperitoneal injection, is delivered to the synapses of motor neurons.

[0260] The experiment was conducted in the same manner as in Example 3, except that the drug was administered by intraperitoneal injection at a dose of 10 mg / kg.

[0261] The results are shown in Figures 8 and 9. Figures 8 and 9 show immunohistochemical staining images 12 hours and 72 hours after administration, respectively. Here, α-BgtX is a marker for acetylcholine receptors on the muscle cell membrane, and SYN1 is a marker for the presynaptic region.

[0262] In the control group using normal rabbit IgG antibody, no signal from normal rabbit IgG antibody was observed at the neuromuscular junction where α-BgtX and SYN1 signals were observed under any time condition (Figures 8A and 9A).

[0263] On the other hand, when an anti-SYT2 N-terminal antibody was used as the antibody, a signal for the anti-SYT2 N-terminal antibody was observed at the neuromuscular junction under all time conditions (Figures 8B and 9B). Furthermore, no abnormalities were observed in the mice or the liver, which is the projection site of the autonomic nerves, after antibody administration. This indicates that the anti-SYT2 N-terminal antibody is delivered to the presynaptic terminal of motor neurons by intraperitoneal injection, similar to when the antibody is administered by tail vein injection.

[0264] <Example 5: Delivery of synaptotagmin 2 antibody to synaptic vesicles of mouse motor neurons by intravenous injection> We investigated whether synaptotagmin 2 antibody, introduced into the body by intravenous injection, is delivered to synaptic vesicles in motor neurons.

[0265] The antibody used for introduction was the same as that used in Example 1. The antibody solution used was prepared by adding antibody to PBS and mixing it until the final concentration was 1 mg / mL.

[0266] The antibody solution was administered to wild-type mice by tail vein injection at a dose of 5 mg / kg. The mice were sacrificed 72 hours after administration, and their calf muscles were harvested and fixed.

[0267] The fixed gastrocnemius muscle was frozen with tissue embedding medium, and 10 μm 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).

[0268] After washing the sections, they were fixed with 2.5% glutaraldehyde, and the nanogold signal was enhanced using the R-gent Se-EM kit (catalog number 500.033; Aurion) and the HQ-silver kit (catalog number 2012; Nanoprobes). Subsequently, the sections were re-fixed with 1% OsO4, dehydrated, and embedded in epong. Ultrathin sections (70 nm thick) were prepared from the embedded sections using an ultramicrotome (catalog number Leica EM UC7; Leica Microsystems). The sections were stained with uranium acetate and lead citrate and observed using a transmission electron microscope (catalog number JEM-1400plus, JEOL).

[0269] The results are shown in Figures 10 and 11. Figures 10 and 11 show transmission electron microscope images of axon terminals of motor neurons (areas enclosed by solid white lines in the figures) and gastrocnemius muscle cells (areas enclosed by dashed black lines in the figures). The black dots in the figures indicate the signal (amplified by silver) that shows the location of the antibody nanogold.

[0270] In the control group using normal rabbit IgG antibody, antibody signals were only sparsely observed at the axon terminals of motor neurons, similar to gastrocnemius muscle cells and other regions (Figure 10A). The signals observed here are thought to be background noise containing artifacts.

[0271] On the other hand, when an anti-SYT2 N-terminal antibody was used, the antibody signal was significantly more pronounced in the nerve axon terminals of motor neurons compared to gastrocnemius muscle cells and other regions (Figure 10B). Therefore, it was found that when an anti-SYT2 N-terminal antibody is used, the antibody is taken up by the nerve axon terminals of motor neurons.

[0272] However, the intracellular membrane structures visible at this magnification mainly consist of mitochondria (arrowheads) and junctional folds (folded structures) in calf muscle cells, and it was not possible to determine from this image alone whether antibodies were being taken up by synaptic vesicles.

[0273] Therefore, samples using an anti-SYT2 N-terminal antibody as the antibody were further enlarged and observed (Figure 11). As a result, it was found that the antibody was incorporated into synaptic vesicles (a and b in Figure 11B) in the axon terminals of motor neurons (b in Figure 11B). From this, it was found that even when the antibody was administered to a living body, the anti-SYT2 N-terminal antibody was delivered into the synaptic vesicles inside the cells in the presynaptic part of motor neuron synapses.

[0274] <Example 6: Delivery of Synaptotagmin 2 Antibody to Mouse Motor Neurons by Intravenous Injection> It was examined whether the synaptotagmin 2 antibody introduced into the living body by intravenous injection was delivered into motor neurons.

[0275] The same antibody as in Example 1 was used as the antibody to be introduced. The antibody solution used was prepared by adding the antibody to PBS and mixing it so that the final concentration was 1 mg / mL.

[0276] The antibody solution was administered to wild-type mice by tail vein injection at a dose of 5 mg / kg.

[0277] The mice were sacrificed 72 hours after administration, and the spinal cords were collected and fixed. The fixed spinal cords were frozen together with a tissue embedding agent, and sections with a thickness of 10 μm were prepared from this frozen tissue block using a cryostat.

[0278] Staining was performed by immunohistochemical staining using the administered antibody as the primary antibody. In the prepared tissue sections, after blocking, a primary antibody reaction was performed using an anti-choline acetyltransferase antibody (α-ChAT antibody; catalog number #NBP1-30052; Novus BioLogicals) as an additional primary antibody. Then, a secondary antibody reaction against each primary antibody was performed, and fluorescence images were obtained. Blocking was performed using a blocking buffer (PBS + 2% normal donkey serum + 1% BSA + 1% fetal bovine serum + 0.02% Triton X-100).

[0279] The same secondary antibody as in Example 3 was used, and fluorescence imaging was performed in the same manner as in Example 3.

[0280] The results are shown in Figures 12-14. Figure 12 shows immunohistochemical staining images observed using objective lenses with 10x magnification (Figure 12) or 40x magnification (Figures 13 and 14). Here, α-ChAT is a marker for choline acetyltransferase in motor neurons.

[0281] When the anterior horn of the spinal cord (left side of the dashed line in Figure 12) was observed using a 10x objective lens, a signal of anti-SYT2 N-terminal antibody was detected in almost all of the cell bodies of motor neurons that showed α-ChAT signaling (Figure 12: arrowhead).

[0282] When the magnification was further increased and observation was performed using a 40x objective lens, the signal of normal rabbit IgG antibody was not observed in the cell bodies of motor neurons where α-ChAT signaling was observed in the control group using normal rabbit IgG antibody (Figure 13).

[0283] On the other hand, when an anti-SYT2 N-terminal antibody was used as the antibody, a signal of the anti-SYT2 N-terminal antibody was observed even in the cell bodies of motor neurons (Figure 14: arrowhead).

[0284] This suggests that intravenously administered anti-SYT2 N-terminal antibody is taken up by synaptic vesicles at synapses located at the neuromuscular junction, and then transported retrogradely through the axon to the cell body.

[0285] <Example 7: Time course observation of synaptotagmin 2 antibody delivered by intravenous injection> The signaling pathway of synaptotagmin 2 antibody, introduced into the body by intravenous injection and delivered into motor neuron cells, was observed over time.

[0286] The antibody used for introduction was the same as that used in Example 1. The antibody solution used was prepared by adding antibody to PBS and mixing it until the final concentration was 1 mg / mL.

[0287] Spinal cord samples were collected from wild-type mice by tail vein injection at a dose of 5 mg / kg.

[0288] The procedure was the same as in Example 7, except that spinal cord samples were collected 6, 24, 72, 120, 168, and 240 hours after administration.

[0289] The results are shown in Figures 15 and 16. Figure 15 shows immunohistochemical staining images collected 6 to 72 hours after administration, and Figure 16 shows immunohistochemical staining images collected 120 to 240 hours after administration.

[0290] The anti-SYT2 N-terminal antibody signal was observed in samples taken 6 hours after administration (Figure 15), and thereafter, the anti-SYT2 N-terminal antibody signal was observed in all samples taken at any time point up to 240 hours after administration (Figures 15 and 16).

[0291] Furthermore, the signal generally tended to become stronger over time. From this, it is presumed that the anti-SYT2 N-terminal antibody is delivered to the cell body within 6 hours of administration and continues to accumulate over time thereafter.

[0292] <Example 8: Verification of pharmacological effects by drug delivery using synaptotagmin 2 antibody> We investigated whether drug-based pharmacological effects could be observed when a drug was delivered into motor neurons using a synaptotagmin 2 antibody, which has been confirmed to be delivered into motor neurons.

[0293] 1. Induction of the presynaptic terminal of motor neurons The experiment was conducted using human motor neurons in which synapse formation was induced using microbeads coated with the extracellular domain of LRRTM2. Cell culture, LRRTM2 bead preparation, and presynaptic induction were performed as described in Example 1.

[0294] 2. Creating a conjugate The drug used was monomethyl auristatin E (MMAE: catalog number #HY-15575; MedChemExpress), a microtubule polymerization inhibitor. The anti-SYT2 N-terminal antibody and the control normal rabbit antibody were the same as those used in Example 1. The MMAE and antibody conjugate was performed using MagicLink. TM The device was fabricated using a kit (Broadpharm). Conjugate fabrication was performed according to the manufacturer's protocol.

[0295] 3. Preparation of the conjugate solution After warming the neuronal culture medium at 37°C for 30 minutes, 4-aminopyridine (Sigma Aldrich) was added to the medium to a final concentration of 100 μM and mixed. The conjugate was then added to a final concentration of 1 μg / mL and mixed. This crude conjugate solution was centrifuged at 200 g for 3 minutes at room temperature, and the supernatant was collected as the conjugate solution.

[0296] Furthermore, as a control group, some samples were treated with MMAE alone instead of the conjugate (number of samples: 13). In this case, the MMAE solution was prepared using the same concentration of MMAE instead of the conjugate, as described above.

[0297] 4. Introduction of Conjugates Conjugates were introduced by changing the culture medium with 100 μL of conjugate solution or MMAE solution on plates in which presynaptic terminals had formed, and then culturing at 37°C for 30 minutes.

[0298] 5. Axonal extension response After introduction, the solution was collected and washed, and the medium was replaced with neuronal medium free of conjugates and MMAE. Then, the cells were cultured for a further 24 hours to allow axonal extension.

[0299] 6. Cell fixation, staining, and observation Cells were fixed in the same manner as in Example 1, and a primary antibody reaction using mouse anti-βIII tubulin (Tuj1) antibody and a secondary antibody reaction using Alexa 555-labeled anti-mouse antibody were performed in the same manner as in Example 1. In this example, staining with anti-SYT2 N-terminal antibody was not performed. Fluorescence images were obtained in the same manner as in Example 1. The sample sizes were as follows: MMAE-only group, 13 samples; control antibody group, 17 samples; SYT2 antibody group, 27 samples.

[0300] 7. Analysis of axonal volume Axonal volume was determined as the sum of the luminance values ​​of the Tuj1 signal in the acquired fluorescence images (= (fluorescence intensity per pixel) × (number of pixels)). The relative axonal volume was calculated by standardizing the results obtained using a conjugate of a control normal rabbit antibody and MMAE to 100%. Luminance was acquired using LAS X software (Leica).

[0301] 8.Statistical analysis Statistical analysis was performed using t-tests with GraphPad Prism9 (GraphPad Software). A significance level of p<0.05 was used.

[0302] The results are shown in Figures 17-19. Figures 17 and 18 are immunocytochemical staining images showing the axons of the control antibody group (Figure 17) using a conjugate of normal rabbit antibody and MMAE, and the SYT2 antibody group (Figure 18) using a conjugate with anti-SYT2 N-terminal antibody. Figure 19 is a graph quantitatively showing the results.

[0303] Under all experimental conditions, axons extended from the neurosphere and presynaptic terminals were induced normally (Figures 17A and 18A).

[0304] MMAE inhibits the polymerization of microtubules, a cytoskeleton crucial for axon elongation and maintenance. Therefore, the stronger the effect of MMAE, the more axon elongation and maintenance are inhibited, and the greater the expected decrease in axon volume.

[0305] In the control antibody group, many thick axons and presynaptic terminals observed as bulges were found even in the distal region far from the neurosphere (Figure 17B). Furthermore, in the proximal region relatively close to the neurosphere, axons were observed to be aligned and running nearly parallel to each other (Figure 17C).

[0306] On the other hand, in the SYT2 antibody group, only a few thin axons were observed in the distal region far from the neurosphere, and presynaptic formation was poor (Figure 18B). Furthermore, in the proximal region relatively close to the neurosphere, the axonal alignment was not as aligned as in the control antibody group, and a disordered axonal alignment was observed (Figure 18C).

[0307] The quantitative results also supported the observations. Compared to the control antibody group ("Cont. IgG-MMAE" in Figure 19), the axonal volume was significantly reduced in the SYT2 antibody group ("α-SYT2 IgG-MMAE" in Figure 19), with the relative axonal volume being approximately 87.15%. Furthermore, the results for the SYT2 antibody group were also significantly reduced compared to the monotherapy group ("MMAE" in Figure 19), with the axonal volume being approximately 82.7% of that of the monotherapy group. No significant difference was observed between the monotherapy group and the control antibody group.

[0308] <Example 9: Confirmation of the relationship between pharmacological effects and nerve activity> By inhibiting the uptake function of synaptic vesicles in nerve cells, we confirmed that the pharmacological effect observed in Example 9 using the anti-SYT2 N-terminal antibody is dependent on synaptic vesicle uptake due to nerve activity.

[0309] The experiment was performed using only the conjugate of anti-SYT2 N-terminal antibody and MMAE. Two groups were used to inhibit synaptic vesicle uptake in nerve cells: a group in which synapse formation was inhibited (synaptic dysplasia group) and a group in which endocytosis was inhibited (endocytosis inhibition group). The experiment was conducted in the same manner as in Example 9, except that motor neurons in the synaptic dysplasia group and the endocytosis inhibition group underwent the following treatments.

[0310] In the group with synaptic dysgenesis, experiments were conducted using microbeads that were not coated with the extracellular domain of LRRTM2 as beads for presynaptic induction.

[0311] In the endocytosis inhibition group, culture was performed at 4°C for 30 minutes after conjugate introduction. The sample size was 3 samples for each group.

[0312] In this example, the relative axonal volume was standardized by setting the axonal volume of the synapse-defective group to 100%.

[0313] The results are shown in Figures 20-23. Figures 20-22 are immunocytochemical staining images showing the axons of the normal firing group (Figure 20), the synaptic dysgenesis group (Figure 21) where presynaptic induction was not performed, and the endocytosis-inhibited group (Figure 22) where endocytosis was inhibited by low-temperature treatment, all under the same conditions as in Example 9. Figure 23 is a graph that quantitatively shows the results.

[0314] Under all experimental conditions, axons extended from the neurosphere (Figures 20-22).

[0315] In the normal firing group, similar to the results shown in Figure 18, there were fewer distal axons, indicating that MMAE inhibited axonal elongation and maintenance (Figure 20). In contrast, in both the synaptic dysplasia group (Figure 21) and the endocytosis inhibition group (Figure 22), axons extended distally, suggesting that the effect of MMAE was limited.

[0316] The quantitative results also supported the observations (Figure 23). In the normal firing group, axonal volume was significantly reduced compared to the synaptic dysplasia group, with relative axonal volume being 81.8%. Furthermore, the results for the normal firing group were also significantly reduced compared to the endocytosis inhibition group, with axonal volume being approximately 80.6% of that of the endocytosis inhibition group. No significant difference was observed between the synaptic dysplasia group and the endocytosis inhibition group.

[0317] These results indicate that inhibiting synaptic vesicle uptake, such as inhibiting synapse formation or endocytosis, almost completely suppresses the pharmacological effects of the conjugate of anti-SYT2 N-terminal antibody and MMAE. Therefore, it is suggested that the pharmacological effects of the conjugate of anti-SYT2 N-terminal antibody and MMAE are mediated by neuronal activity, particularly endocytosis at synapses.

[0318] <Example 10: Verification of pharmacological effects when targeting other membrane proteins> Similar to the case using anti-SYT2 N-terminal antibodies, we also investigated whether antibodies against other membrane proteins present in synaptic vesicles could be used for drug delivery.

[0319] The experiment was conducted in the same manner as in Example 9, except for the antibody used. The antibody used is as follows: Anti-SYT2 N-terminal antibody: Polyclonal rabbit purified antibody SYT2 lumenal domain (Catalog No. 105 223; Synaptic Systems); Sample size: 27; Anti-SYP antibody: Anti-Synaptophysin Antibody Major synaptic vesicle protein p38, SYP (Catalog number ANR-013; alomone labs; Epitope: SEQ ID NO: 21); Sample size: 20; Anti-SYNGR1 antibody: Synaptogyrin 1 Antibody - BSA Free (Catalog No. NBP1-77371; Novus Biologicals; Epitope: SEQ ID NO: 17); Sample size: 20; Anti-SYT1 antibody: Synaptotagmin1 antibody luminal domain (Catalog number 105 103; Synaptic Systems; Epitope: SEQ ID NO: 24); Sample size: 15; Anti-SV2A antibody: SV2A Polyclonal Antibody (Catalog number BS-2407R; Bioss Antibodies; Epitope: SEQ ID NO: 13); Sample size: 14

[0320] In this example, the relative axonal length was calculated by standardizing the results obtained using a conjugate of control normal rabbit antibody and MMAE to 100%.

[0321] The results are shown in Figure 24. Figure 24 shows the relative axonal volume when conjugates using each antibody are introduced.

[0322] Even when membrane proteins present in synaptic vesicles other than anti-SYT2 N-terminal antibodies were used, axonal volume was significantly reduced, with relative axonal volume ranging from 87.2% to 90.7% (Figure 24). No significant difference in relative axonal volume was observed depending on the type of antibody used.

[0323] Therefore, it was confirmed that drugs can be delivered to motor neurons by using antibodies against membrane proteins present in synaptic vesicles, regardless of the type of membrane protein being targeted. Furthermore, this suggests that it is possible to confer to target cells the effects based on those drugs.

[0324] All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims

1. A targeting agent for motor neurons containing an antibody capable of binding to the intravesicular domain of membrane proteins present in synaptic vesicles of motor neurons.

2. The targeting agent according to claim 1, further comprising a labeled substance and / or a physiologically active substance.

3. The targeting agent according to claim 2, comprising a conjugate of the antibody and / or the labeling substance and / or the physiologically active substance.

4. The targeting agent according to claim 1, wherein the membrane protein is a human-derived protein.

5. The targeting agent according to claim 1, wherein the membrane protein comprises any one protein selected from the group consisting of synaptotagmin 2, synaptic vesicle glycoprotein 2A, synaptogyrin 1, synaptophysin, and synaptotagmin 1.

6. The targeting agent according to claim 1, wherein the intravesicular domain is a domain containing four or more amino acids.

7. The targeting agent according to claim 5, wherein the intravesicular domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7-12, 15, 16, 19, 20, and 23; an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7-12, 15, 16, 19, 20, and 23; or an amino acid sequence having 90% or more sequence identity with the amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 7-12, 15, 16, 19, 20, and 23.

8. The targeting agent according to claim 2, wherein the labeling substance is a fluorescent molecule.

9. The targeting agent according to claim 2, comprising the labeling substance which is a motor neuron visualization agent.

10. The targeting agent according to claim 2, wherein the physiologically active substance is one or more selected from the group consisting of synapse formation promoters, synapse maintenance agents, muscle strengthening agents, and nerve cell function modifying agents.

11. The targeting agent according to claim 1, which is taken up into cells by endocytosis.

12. A pharmaceutical composition comprising the targeting agent described in claim 2.

13. A method for targeting motor neurons with a labeled substance and / or a bioactive substance, comprising the steps of: contacting motor neurons with a targeting agent according to any one of claims 2 to 11 and / or a pharmaceutical composition according to claim 12; and delivering the targeting agent and / or the pharmaceutical composition to the synapses of the motor neurons.

14. A method for visualizing motor neurons, comprising the steps of: bringing the motor neuron visualization agent described in claim 9 into contact with motor neurons; delivering the motor neuron visualization agent to the synapse of the motor neurons; and detecting the signal of the labeling substance.

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