Targeting agent

Abstract

The present invention addresses the problem of providing a means for targeting a substance to central nervous system cells when the substance is administered into peripheral blood.　Provided is a targeting agent targeting central nervous system cells that express a membrane protein existing in a synaptic vesicle of a motor neuron, said targeting agent comprising a conjugate of an antibody able to bind to an intravesicular domain of the membrane protein and a labeling substance and / or a bioactive substance and being incorporated into a secondary motor neuron.

Description

Targeting agent

[0001] The present invention relates to a targeting agent for central nervous system cells, a central nervous system cell visualization agent, and compositions containing the same, as well as methods for targeting, methods for visualizing central nervous system cells, and methods for preventing or treating a condition or disease using the same.

[0002] The nervous system, composed of the central and peripheral nervous systems, regulates various emotions, muscle functions, and the functions of internal organs. These nerve functions can decline due to nerve damage, disease, and aging, which can impair physical and mental health and have a significant impact on daily life. Therefore, maintaining and improving nerve function is an extremely important issue, as it directly impacts the quality of life (QOL).

[0003] The central and peripheral nervous systems are each composed of nerve cells, which exchange signals with each other via synapses. A synapse is a junction containing a gap formed between the axon terminal of one nerve cell (presynaptic terminal) and the dendrite of another nerve cell or cells of skeletal muscle or organs (postsynaptic terminal). Signals are transmitted when chemical substances released from the presynaptic terminal bind to receptors present on the postsynaptic terminal. Synapse formation is triggered by the interaction of specific membrane proteins expressed in the presynaptic and postsynaptic terminals.

[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.

[0006] Japanese Patent Publication No. 2012-092048, Japanese Patent Publication No. 2008-143867, WO2021 / 006075

[0007] The inventors previously developed a technology for delivering the above-mentioned compounds and drugs to target motor neurons via controlled transport (WO2023 / 210585).

[0008] However, central and peripheral nerve cells are interconnected, and the presence and degree of nerve stimulation input and output affect the properties of nerve cells. Therefore, in neurological diseases such as the neurodegenerative diseases mentioned above, even if symptoms are mainly present in the peripheral nerves, abnormalities are almost always present in the central nerve cells as well.

[0009] For example, in the early stages of a disease, treating peripheral nerve cells may lead to the recovery of central nervous system cells that communicate with them. However, as the disease progresses and symptoms worsen, it may become necessary to treat central nervous system cells in addition to peripheral nerve cells.

[0010] Therefore, the object of the present invention is to provide a means for targeting not only motor neurons but also central nervous system cells with a substance, and a means for visualizing the targeting site.

[0011] The inventors focused on the fact that certain proteins expressed on the membrane of synaptic vesicles are also expressed in central nervous system cells. As a result of diligent research to solve the above problem, they found that by using an antibody that binds to the intravesical domain (N-terminal portion) of synaptotagmin 2, and observing the effects over a long period of time after administering an excessive amount, it is possible to target the desired substance in central nervous system cells based on the properties unique to motor neurons, thus completing the present invention.

[0012] In other words, the present invention encompasses the following: [1] A targeting agent for central nervous system cells expressing a membrane protein, comprising a conjugate of an antibody capable of binding to the intravesicular domain of a membrane protein present in synaptic vesicles of motor neurons and a labeling substance and / or a physiologically active substance, which is taken up into secondary motor neurons. [2] The targeting agent according to [1], wherein the membrane protein is a human-derived protein. [3] The targeting agent according to [1] or [2], 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. [3-1] The targeting agent according to [1] or [2], wherein the membrane protein comprises any one protein selected from the group consisting of synaptotagmin 2, synaptic vesicle glycoprotein 2A, synaptogyrin 1, synaptogyrin 3, synaptophysin, and synaptotagmin 1. [4] The targeting agent according to any one of [1] to [3-1], wherein the intravesicular domain is a domain containing four or more amino acids. [5] The targeting agent according to [3], 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. [6] The targeting agent according to any one of [1] to [5], wherein the labeling substance is a fluorescent molecule. [7] The targeting agent according to any one of [1] to [6], comprising the labeling substance, which is a neuronal cell visualization agent. [8] The targeting agent according to any one of [1] to [7], wherein the physiologically active substance is one or more selected from the group consisting of synapse formation promoters, synapse maintenance agents, and nerve cell function modifiers. [9] The targeting agent according to any one of [1] to [8], which is taken up into central nervous system cells by endocytosis.

[10] A pharmaceutical composition comprising the targeting agent according to any one of [1] to [9].

[11] A method for targeting a labeling substance and / or a bioactive substance to central nervous cells expressing the membrane protein, comprising the steps of: contacting a secondary motor neuron with a targeting agent according to any one of [1] to [9] and / or a pharmaceutical composition according to

[10] , which includes a conjugate; delivering the conjugate into cerebrospinal fluid; and causing the labeling substance and / or bioactive substance to be taken up into the cells of the central nervous cell.

[12] A method for visualizing central nervous cells expressing the membrane protein, comprising the steps of: contacting a secondary motor neuron with a visualization agent according to [7], which includes a conjugate; delivering the conjugate into cerebrospinal fluid; causing the labeling substance to be taken up into the cells of the central nervous cell by the secondary motor neuron; and detecting the signal of the labeling substance. This specification includes the disclosures of Japanese Patent Application No. 2024-176336, which forms the basis of the priority of this application.

[0013] According to the targeting agent and targeting method of the present invention, a desired substance can be targeted to central nervous system cells using an antibody. By using an antibody, the desired substance can be delivered to central nervous system cells (e.g., synaptic vesicles within those cells). If the substance is a physiologically active substance or a therapeutic agent, symptoms and / or diseases caused by abnormalities in central nervous system cells can be treated. Furthermore, if the substance is a labeled substance, central nervous system cells can be visualized.

[0014] Figure 1 shows the signal of anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) in the coronal section of a mouse brain hemisphere 240 hours after intravenous administration of synaptotagmin 2 antibody. In the figure, the shaded areas indicate the locations of major ventricles and nerve bundles. Figure 2 shows the signal of anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) in the coronal section of a mouse brain hemisphere 240 hours after intravenous administration of synaptotagmin 2 antibody. In the figure, the shaded areas indicate the locations of major ventricles and nerve bundles. Figure 3 shows the signal of anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) in the coronal section of a mouse brain hemisphere 240 hours after intravenous administration of synaptotagmin 2 antibody. In the figure, the shaded areas indicate the locations of major ventricles and nerve bundles. Figure 4 shows the signal of anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) in coronal sections of mouse brain hemispheres 240 hours after intravenous administration of synaptotagmin 2 antibody. In the figure, the shaded areas indicate the locations of major ventricles and nerve bundles. Figure 5 shows the signal of anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) in coronal sections of mouse brain hemispheres 240 hours after intravenous administration of synaptotagmin 2 antibody. In the figure, the shaded areas indicate the locations of major ventricles and nerve bundles. Figure 6 shows the signal of anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) in coronal sections of mouse cerebellum and brainstem 240 hours after intravenous administration of synaptotagmin 2 antibody. Figure 6A is a fluorescence image of the cerebellum, and Figure 6B is a fluorescence image of the brainstem. In the figures, the shaded areas indicate the locations of major ventricles and nerve bundles. In Figure 6B, the area enclosed by the white dashed line indicates the location of facial motor nuclei cells where the cell bodies of secondary motor neurons are located. Figure 7 shows the radioactive isotopes of Example 2. 89This figure shows the results of the Zr-labeled antibody administration experiment. Figure 7A shows the blood antibody level 4 hours after administration of the labeled antibody, and Figure 7B shows the blood antibody level 78-79 hours after administration of the labeled antibody. In the figure, "Cont." indicates the results of the control antibody, anti-keyhole limpet hemocyanin antibody, and "αSYT2-1" indicates the results of the αSYT2-1 antibody in Table 4. In Figure 7B, "**" indicates that p < 0.01. Figure 8 shows fluorescence images of the neuromuscular junction in the antibody administration experiment of Example 3. Figure 8A shows the fluorescence image of the neuromuscular junction when anti-VGAT antibody was administered, and Figure 8B shows the fluorescence image of the neuromuscular junction when anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) was administered. In the figure, arrowheads indicate neuromuscular junctions where no signal for anti-VGAT antibody was observed, and arrows indicate neuromuscular junctions where a signal for anti-SYT2 N-terminal antibody was observed. In the figure, the scale bar in the lower right indicates 50 μm. Figure 9 shows fluorescence images of the spinal cord in the antibody administration experiment of Example 3. Figure 9A shows the fluorescence image of the spinal cord when anti-VGAT antibody was administered, and Figure 9B shows the fluorescence image of the spinal cord when anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) was administered. In the figure, the dashed line shows the outline of the spinal cord, and the dotted line shows the location of the anterior horn of the spinal cord. In the figure, the scale bar in the lower right indicates 200 μm. Figure 10 shows fluorescence images of the anterior horn of the spinal cord in the antibody administration experiment of Example 3. Figure 10A shows the fluorescence image of the anterior horn of the spinal cord when anti-VGAT antibody was administered, and Figure 10B shows the fluorescence image of the anterior horn of the spinal cord when anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) was administered. In the figure, arrowheads indicate neuromuscular junctions where no signal for anti-VGAT antibody is observed, and arrowheads indicate neuromuscular junctions where a signal for anti-SYT2 N-terminal antibody is observed. In the figure, the scale bar in the lower right represents 100 μm. Figure 11 is a fluorescence image of the sagittal section of the brain from the antibody administration experiment of Example 3. Figure 11A shows a fluorescence image of the sagittal section of the brain after administration of anti-VGAT antibody, and Figure 11B shows a fluorescence image of the sagittal section of the brain after administration of anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody). In the figure, dashed lines indicate the contour of the sagittal section of the brain, and in Figure 11B, dotted lines indicate the brain regions where the main anti-SYT2 N-terminal antibody signal was observed.In the figure, the scale bar in the lower right represents 1 mm. Figure 12 is a fluorescence image of the administration experiment of anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) in Example 4. Figure 12A shows a fluorescence image of the spinal cord anterior horn, and Figure 12B shows a fluorescence image of the hippocampus. In the figure, the arrows indicate spinal cord anterior horn cells where the signal of the anti-SYT2 N-terminal antibody is observed, and the dashed lines indicate the regions where the signal of the anti-SYT2 N-terminal antibody is observed. In the figure, the scale bar represents 100 μm. Figure 13 is a figure showing the results of a malat1 expression suppression experiment using anti-synaptotagmin 2 intravesicular domain (N-terminal) antibody (anti-SYT2 N-terminal antibody) in Example 5. In the figure, "Cont." indicates the results when a control oligonucleotide was used, and "ASO" indicates the results when an antisense oligonucleotide against malat1 was used. In the figure, the error bars indicate the standard deviation, and "#" indicates that p < 0.1.

[0015] The present invention will be described in detail below. <Targeting Agent> The present invention relates to a targeting agent for central nervous system cells (referred to as "the targeting agent of the present invention") comprising an antibody capable of binding to the intravesicular domain of a membrane protein present in synaptic vesicles of motor nerve cells (referred to as "intravesicular domain antibody") and a conjugate of a labeled substance and / or a physiologically active substance (referred to as "the conjugate of the present invention").

[0016] The antibody contained in the targeting agent of the present invention is capable of binding to the intravesicular domain of a membrane protein present in the synaptic vesicles of motor neurons. Upon contact with secondary motor neurons, it binds to antigens present on the cell membrane and is taken up into the secondary motor neurons along with a labeling substance and / or a bioactive substance. Subsequently, the antibody is released from the secondary motor neurons into the interstitial space and delivered into the cerebrospinal fluid. Since this membrane protein is also expressed in some central nervous system cells, the targeting agent of the present invention is taken up by central nervous system cells by binding to antigens on the cell membranes of those central nervous system cells. As a result, the targeting agent of the present invention is targeted to those central nervous system cells.

[0017] 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. The membrane proteins described herein are membrane proteins present in synaptic vesicles of motor neurons and expressed in some central nervous system cells. The expression mode, such as the expression site of the membrane protein in central nervous system cells, is not particularly limited, but for example, it is present on the membrane of a synaptic vesicle.

[0018] The type of membrane protein in the targeting agent of the present invention may be any protein containing an intravesicular domain, and is not particularly limited, but is preferably a transmembrane protein.

[0019] 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.

[0020] In this specification, "lumen of synaptic vesicle" refers to the space located on the opposite side of the cytoplasm within a synaptic vesicle.

[0021] 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.

[0022] First, let's explain using synaptotagmin 2 as a membrane protein as an example. The present invention relates to a targeting agent for central nervous system cells, comprising a conjugate of an antibody capable of binding to the intravesicular domain (N-terminal portion) of synaptotagmin 2 (referred to as "anti-SYT2 N-terminal antibody") and a labeling substance and / or a physiologically active substance.

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

[0026] Exemplary mouse synaptotagmin 2 is a protein consisting of 422 amino acids whose amino acid sequence is represented by SEQ ID NO: 2. The positions of each domain are, in SEQ ID NO: 2, the luminal domain is the region represented by the amino acid sequence from position 1 to position 60, the transmembrane domain is the region represented by the amino acid sequence from position 61 to position 87, and the cytoplasmic domain is the region represented by the amino acid sequence from position 88 to position 422.

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

[0028] In the present specification, the "luminal domain of synaptotagmin 2" refers to all or part of the luminal domain present in the N-terminal portion of synaptotagmin 2. As the whole of the luminal domain, for example, the region represented by the amino acid sequence from position 1 to position 62 (SEQ ID NO: 3) in SEQ ID NO: 1 and the region represented by the amino acid sequence from position 1 to position 60 (SEQ ID NO: 4) in SEQ ID NO: 2 are included. Also, as part of the luminal domain, for example, regions represented by partial sequences of any length in SEQ ID NOs: 3 and 4 are included. More specifically, for example, the regions represented by the amino acid sequences from position 1 to position 11 (both identical to SEQ ID NO: 5) in SEQ ID NOs: 3 and 4 and 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: 5 are included. Here, "a plurality" refers to a number of 2 or more, for example, 2 to 6, 2 to 5, 2 to 4, or 2 to 3, preferably 2.

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

[0030] As specific membrane proteins in the targeting agent of the present invention, in addition to synaptotagmin 2, for example, synaptotagmin family, major facilitator superfamily, synaptic vesicle glycoprotein 2 family, synaptogyrin family, synaptophysin / synaptobrevin family, synaptobrevin family, vesicular amine transporter family, solute carrier family 5, vesicular glutamate transporter family, amino acid / polyamine transporter family, secreted carrier-associated membrane protein family, glutamate transporter subfamily, autophagy-related protein 9 family, sugar transporter family, vacuolar-type ATPase subunit S1 family, a protein belonging to any one family selected from the group consisting of them can be mentioned.

[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, 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 proteins in the targeting agent of the present invention include 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 intravesicular 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 in accordance with the description 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 the organization of vesicle membrane components, targeting of vesicles to the cell membrane, and regulation of synaptic plasticity. Synaptophysin is also known by other names such as MRX96, tumor synaptic vesicle protein P38, MRXSYP, and XLID96. An exemplary synaptophysin is a human-derived protein 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, the protein region of synaptophysin includes the region indicated by the amino acid sequence from positions 178 to 190 of SEQ ID NO: 18 (SEQ ID NO: 21: CRQTGNTCKELRD) and 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: 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 term "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. Vesicular acetylcholine transporters are 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 number 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 position 6 in SEQ ID NO: 36 (sequence: MAFHVE; SEQ ID NO: 37), This includes the region indicated by the amino acid sequence from position 70 to 81 (sequence number: GTAEAVYVPGYG; SEQ ID NO: 38), the region indicated by the amino acid sequence from position 147 to 164 (sequence number: GEMFWAAAIFSALGATISVIIDVDMHIS; SEQ ID NO: 39), the region indicated by the amino acid sequence from position 213 to 237 (sequence number: ADIGFTAVHAKYQKPWLGTVDSSEV; SEQ ID NO: 40), the region indicated by the amino acid sequence from position 296 to 317 (sequence number: ASTDWNQTAYGLPDPKTTEEAD; SEQ ID NO: 41), the region indicated by the amino acid sequence from position 398 to 406 (sequence number: LTKTVYGLW; SEQ ID NO: 42), and the region indicated by the amino acid sequence from position 457 to 481 (sequence number: 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 cotransporter 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, "intravesicular domain of vesicular glutamate transporter 3" refers to all or part of the intravesicular domain of vesicular glutamate transporter 3. The entire intravesicular domain includes, for example, the region 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] 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 exemplary synaptic vesicle glycoprotein 2B is a human-derived protein consisting of 683 amino acids, represented by the amino acid sequence SEQ ID NO: 61.

[0051] 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.

[0052] 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.

[0053] 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, for example, the region indicated by a subsequence of any length in Sequence IDs 68 to 72. The length of the subsequence is in accordance with the description for the anti-SYT2 N-terminal antibody.

[0054] 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 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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 SEQ ID NO: 77.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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 represented 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.

[0068] 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 that participates in autophagy by modifying the phospholipid composition on the autophagosome membrane and mediating its expansion. Autophagy-Related Protein 9A is also known by other names such as APG9L1, FLJ22169, and MATG9. An exemplary Autophagy-Related Protein 9A is a human-derived protein consisting of 839 amino acids, represented by the amino acid sequence number 95.

[0069] 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 partial sequence of any length in SEQ ID NOs: 96 and 97. The length of the partial sequence is as described for the anti-SYT2 N-terminal antibody.

[0070] 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.

[0071] 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.

[0072] 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 acidification of 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.

[0073] 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 may include, 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. In addition, 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.

[0074] 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.

[0075] 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.

[0076] 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 central nervous system cells, particularly central nervous system 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] "Central nervous system cells" refers to the group of nerve cells that make up the brain and spinal cord. In this specification, central nervous system cells refer specifically to nerve cells that have their axon terminals in the central nervous system, among the nerve cells that make up the brain and spinal cord. The central nervous system cells targeted in this specification are central nervous system cells that express membrane proteins to which antibodies contained in the targeting agent or conjugate of the present invention can bind. Preferably, the central nervous system cells in this specification are nerve cells that have their axon terminals and cell body in the central nervous system, or nerve cells that have their entire body in the central nervous system. Information on central nervous system cells that express the target membrane protein or information on membrane proteins expressed by the target central nervous system cells is readily available from various databases. Examples of such databases include Uniprot and GeneCards. (R) Examples include the Allen Brain Atlas and The Human Protein Atlas.

[0082] The specific types of central nervous system cells are not limited. Examples include cerebral cortical cells, hippocampal cells, basal ganglia cells, thalamic cells, hypothalamic cells, midbrain cells, pontine cells, medullary cells, and cerebellar cells. Specifically, for example, in the case of synaptotagmin 2, examples include inhibitory (GABAergic) central nervous system cells and parvalbumin-positive GABAergic central nervous system cells. The areas in which these central nervous system cells reside are not particularly limited, but in the case of synaptotagmin 2, for example, primary motor cortex cells, supplementary motor cortex cells, somatosensory cortex cells, insular cortex cells, incomplete insular cortex cells, agranulosular cortex cells, piriform lobe cortex cells, cingulate gyrus cortex cells, caudal interstitial nucleus cells of the medial longitudinal fasciculus, diagonal band cells, medial septal nucleus cells, ventral pallidal nucleus cells, magnocellular preoptic nucleus cells, lateral preoptic nucleus cells, hippocampal pyramidal cell layer cells, dentate gyrus granulosa cells, habenula nucleus cells, lateral geniculate nucleus cells, dorsolateral tegmental nucleus cells, dorsolateral nucleus cells, posterolateral nucleus cells, posterior thalamic nucleus cells, medial inferior thalamic nucleus cells, ventromedial hypothalamic nucleus cells, anterior uncinate gyrus cells, parauncinate gyrus cells, uncinate gyrus cells, cerebellar granulosa cells, perifacial zone cells, trigeminal spinal tract nucleus cells, inferior frontal gyrus cells (orbital pars cells, pars trigonum cells, etc.), ventrolateral prefrontal cortex cells, anterior cingulate gyrus cells (anterior subgenicular cells, etc.), frontopolar cortical cells, prelimbic cortical cells, subcallosal cells, superior frontal gyrus cells, lateral orbitofrontal gyrus cells, retroorbital frontal gyrus cells, dorsolateral prefrontal cortex cells, endodorsal cells Examples include lateral prefrontal cortex cells, paracentral lobule (anterior, posterior, etc.) cells, precentral gyrus (dorsal, middle, ventral, etc.) cells, postcentral gyrus (dorsal, middle, ventral, etc.) cells, dorsal premotor cortex cells, ventral premotor cortex cells, occipital cortex cells, angular gyrus cells, inferior parietal lobule cells, superior parietal lobule cells, parietal insular cortex cells, and the like.

[0083] In this specification, "central" refers to the cerebral circulation side with respect to the blood-brain barrier. Conversely, in this specification, "peripheral" refers to the systemic circulation side with respect to the blood-brain barrier.

[0084] In this specification, "cerebrospinal fluid" refers to the tissue fluid of the brain and spinal cord. In this specification, cerebrospinal fluid includes not only the cerebrospinal fluid filling the ventricles that constitute the cerebral circulation, but also a broad range of intercellular fluids of the brain parenchyma and spinal cord parenchyma.

[0085] 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.

[0086] According to the targeting agent of the present invention, a desired substance (labeled substance and / or bioactive substance) can be targeted to central nervous system cells (e.g., axon terminals, axons, axonal papillae, cell bodies, dendrites, etc. of central nervous system cells) via central nervous system cell synapses.

[0087] 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 or organs (in the case of the peripheral nervous system).

[0088] 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").

[0089] 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.

[0090] 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.

[0091] 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 of central nervous system cells, is taken up into synaptic vesicles by endocytosis, and can then be delivered to the cell body of the central nervous system cell. Therefore, the targeting agent of the present invention makes it possible to target a desired substance (labeled substance and / or bioactive substance) into the interior of central nervous system cells, particularly into the cell body of the central nervous system cell.

[0092] 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.

[0093] 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.

[0094] 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.

[0095] Examples of antibodies available in this specification include antibodies containing the CDR sequences shown in Tables 1-3.

[0096]

[0097]

[0098]

[0099] The amino acid sequence of the variable region of each antibody is not particularly limited, as long as it has the same CDR sequence as the antibody names shown in Tables 1 to 3. For example, it may consist of the amino acid sequences shown in Tables 4 to 6; amino acid sequences having 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequences shown in Table 2; or amino acid sequences in which one or more amino acids are substituted, deleted, and / or added. The preparation of an antibody functionally equivalent to a given antibody can be carried out by methods well known in this field, such as site-directed mutagenesis.

[0100]

[0101]

[0102]

[0103] In this specification, "amino acid substitution" refers to substitutions between the 20 amino acids that make up natural proteins. Amino acid substitutions are not particularly limited, but may be conservative substitutions. Conservative substitutions refer to substitutions within a group of conservative amino acids that have similar properties such as charge, side chain, polarity, and aromaticity. Examples include substitutions within the group of uncharged polar amino acids with low-polarity side chains (Gly, Asn, Gln, Ser, Thr, Cys, Tyr), branched-chain amino acids (Leu, Val, Ile), neutral amino acids (Gly, Ile, Val, Leu, Ala, Met, Pro), neutral amino acids with hydrophilic side chains (Asn, Gln, Thr, Ser, Tyr, Cys), acidic amino acids (Asp, Glu), basic amino acids (Arg, Lys, His), and aromatic amino acids (Phe, Tyr, Trp).

[0104] In vivo, after the formation of an antibody against a certain antigen, a process called "affinity maturation" is known to produce an antibody with improved affinity for the antigen compared to the initial antibody. Therefore, based on the amino acid sequence of the antibody specifically disclosed above, it is possible to obtain an antibody with activity equivalent to or better than the disclosed antibody, specifically an antibody with improved binding affinity to the intravesicular domain of membrane proteins such as synaptotagmin 2, and improved functionality as a targeting agent for substance delivery. These modifications can occur not only within the framework region and constant region, but also within the CDR region.

[0105] The method for obtaining the antibody contained in the targeting agent of the present invention is not particularly limited. For example, it can be obtained as a monoclonal antibody by known methods after immunizing a non-human mammal with the intravesicular domain of a membrane protein identified as an antigen. The antibody of the present invention can also be obtained by synthesis using genetic engineering methods or chemical synthesis means, based on the amino acid sequence information of the antibody of the present invention whose activity has been demonstrated or the base sequence information of the polynucleotide encoding the antibody.

[0106] When producing antibodies using genetic engineering techniques, polynucleotides encoding heavy and light chains can be introduced into appropriate host cells for expression, and the antibody can be obtained as a recombinant protein. In this case, the polynucleotides may be DNA or RNA, and the means of introduction into the host cells can be any method used in this field. Viral vectors, plasmid vectors, phage vectors, etc., can be used as vectors for introducing polynucleotides into host cells. Host cells can be, for example, bacteria such as E. coli, yeast, insect cells, animal cells, etc. Here, the polynucleotides encoding the heavy and light chains may be introduced into separate vectors or ligated into the same vector.

[0107] 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.

[0108] 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.

[0109] Instead of antibodies as described herein, any molecule capable of binding to the target molecule can be used as a targeting agent or central nervous system cell 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.

[0110] The targeting agent of the present invention further comprises an antibody capable of binding to the intravesicular domain (N-terminal portion) of a membrane protein, including an antibody capable of binding to the intravesicular domain (N-terminal portion) of synaptotagmin 2, as well as a desired substance (labeling substance and / or physiologically active substance).

[0111] 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, 3 H, 125 I, 89 Examples of reagents include those containing Zr. 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.

[0112] 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 central nervous system 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, or nerve cell function modifiers can be used as bioactive substances.

[0113] "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.

[0114] The desired substance (labeled substance and / or bioactive substance) contained in the targeting agent of the present invention is included in the form of a conjugate covalently linked to an antibody capable of binding to the intravesicular domain of a membrane protein, including an antibody capable of binding to the intravesicular domain of synaptotagmin 2.

[0115] 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).

[0116] 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, for example, (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).

[0117] 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).

[0118] Specific synapse formation promoting agents and synapse maintenance 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.

[0119] 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).

[0120] 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 other microtubule polymerization inhibitors (including auristatin-based drugs such as monomethyl auristatin E (MMAE), monomethyl auristatin F, and auristatin PE), as well as other cytoskeletal modifiers.

[0121] 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.

[0122] Since these drugs act on central nervous system cells, any drug that exhibits an effect on central nervous system cells is acceptable. For example, drugs that have a central nervous system-specific effect, drugs that exhibit an effect on central nervous system cells and do not have a harmful effect on motor nerve cells (either they do not act on motor nerve cells or they have a beneficial effect on motor nerve cells), etc., can be suitably used.

[0123] 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, or they may be indirectly linked via a linker or the like.

[0124] 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.

[0125] Furthermore, multiple labeling substances and / or physiologically active substances may be included, as long as they do not impair the functions of each other. In this case, for example, multiple identical substances may be included, or one or more different substances may be included.

[0126] In this 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 a cleavable linker or a linker that is not cleaved after transport to the synapse.

[0127] 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.

[0128] 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. For example, 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 the labeled substance and / or the bioactive substance are 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 physiologically active substance, the overall particle size when the labeling substance and / or a physiologically active substance is bound to the targeting agent can be within the range described above.

[0129] The conjugate or targeting agent of the present invention does not need to have a special configuration to cross the blood-brain barrier.

[0130] The conjugate or targeting agent of the present invention, once taken up by secondary motor neurons, acts on central nervous system cells by being partially delivered into the cerebrospinal fluid.

[0131] The targeting effect on central nervous system cells can be determined, for example, by administering the conjugate or targeting agent of the present invention, which contains a bioactive substance, into the peripheral blood of 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 of the present invention on the central nervous system cells of the target. 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.

[0132] 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).

[0133] Therefore, whether or not a conjugate or targeting agent is taken up by endocytosis can also be determined by examining whether or not the test substance is localized to the presynaptic region induced by co-culture of nerve cells and microbeads.

[0134] "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.

[0135] <Conjugate> The present invention relates to a conjugate of an antibody capable of binding to the intravesicular domain (N-terminal portion) of a membrane protein present in 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 physiologically active substance. Similar to the targeting agents described above, the conjugate of the present invention is taken up into the synaptic vesicles of secondary motor neurons and central nervous system cells and delivered to the central nervous system cells.

[0136] The drug-antibody ratio (DAR) in a conjugate can be set appropriately depending on the purpose and is not particularly limited. For example, it can be 1 or greater, 1.5 or greater, 2 or greater, 2.5 or greater, etc. Alternatively, it can be 10 or less, 9 or less, 7 or less, 5 or less, 4 or less, 3 or less, etc. Specific ranges for DAR values ​​include, for example, 1 to 10, 1 to 9, 1.5 to 7, 1.5 to 5, 2 to 4, 2 to 3, etc.

[0137] <Visualizing agent for central nervous system cells or their synapses> The present invention provides a targeting agent (referred to as "the visualization agent of the present invention") which is a visualizing agent for central nervous system cells or their synapses.

[0138] The visualization agent of the present invention is a targeting agent containing a labeling substance for use in visualizing central nervous system cells or their synapses.

[0139] "Visualizing central nervous system cells" refers to making all or part of central nervous system cells detectable. When visualizing a part of central nervous system cells, the visualized part may be random or a predetermined part. For example, synapses and / or nerve fibers can be visualized as predetermined parts. In that case, the visualization agent of the present invention can be used as a nerve fiber visualization agent. "Visualizing nerve fibers" refers to making axons and / or dendrites 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 axon terminals, axons, axonal papillae, cell bodies, dendrites, etc., of central nervous system cells.

[0140] The visualization agent of the present invention can be used in vivo. Detection of the signal of the labeled substance can be performed while the central nervous system cells are alive or after the central nervous system cells have been fixed. Labeled substances suitable for detection in the living state of central nervous system cells 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.

[0141] The visualization agent of the present invention may be used for any application, for example, to visualize the number, size, or location of central nervous system cells or their nerve fibers, or to visualize tissue in surgical procedures or diagnostics.

[0142] 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.

[0143] 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.

[0144] <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.

[0145] 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.

[0146] 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.

[0147] 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.

[0148] When the composition or pharmaceutical composition of the present invention contains a synapse formation promoter or synapse maintenance 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 purpose of improving nerve function.

[0149] In this specification, “nerve damage” means 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, tumors, and diseases.

[0150] 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.

[0151] Frailty is a known condition that arises from 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, often due to the coexistence of multiple chronic diseases, resulting in a weakened physical and mental state. 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. Examples of chronic diseases include hypertension, heart disease, cerebrovascular disease, diabetes, respiratory diseases, and malignant tumors.

[0152] 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 frailty or are at high risk of having frailty.

[0153] In the present invention, examples of diseases include neurological diseases. In this specification, "neurological disease" refers to a disease caused by a disorder of the central nervous system or other nerves, and for example, refers to one or more diseases selected from the group consisting of: Alzheimer's disease, Parkinson's disease, Lewy body dementia, frontotemporal lobar degeneration, progressive supranuclear palsy, corticobasal degeneration, Huntington's disease, dystonia, prion disease, mitochondrial disease, acanthocyte chorea, adrenoleukodystrophy, multiple system atrophy, spinal cord atrophy This includes conditions such as neurodegeneration, 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, neuropathy (multifocal motor neuropathy, etc.), muscular dystrophy, myopathy, congenital myasthenic syndrome, hereditary periodic paralysis, myasthenia gravis, and Lambert-Eaton syndrome. When targeting the aforementioned conditions such as muscular dystrophy, myopathy, congenital myasthenic syndrome, hereditary periodic paralysis, myasthenia gravis, and Lambert-Eaton syndrome, the pharmaceutical composition of the present invention can treat disease-related central nervous system cells in conjunction with motor nerve cells.

[0154] Epilepsy is a disease characterized primarily by seizures (epileptic seizures) that occur in a limited number of nerve cells due to excessive electrical excitation. These seizures involve motor nerve cells, sensory nerve cells, autonomic nerve cells, and central nervous system cells associated with higher brain functions. In this specification, epilepsy includes focal epilepsy (partial epilepsy), generalized epilepsy, generalized focal epilepsy, and epilepsy of unknown type. Therefore, the compositions or pharmaceutical compositions of the present invention that contain neuronal function modifiers that cause changes (e.g., inhibition) in the electrophysiological properties of nerve cells (such as the properties of conduction and transmission of stimuli), can be used in particular to improve or prevent the symptoms of epilepsy.

[0155] Schizophrenia is a mental disorder characterized by hallucinations, delusions, negative symptoms, cognitive impairment, and impaired brain function. Diagnosis can be made based on the World Health Organization's (WHO) International Classification of Diseases, 10th Revision (ICD-10) or the American Psychiatric Association's Diagnostic and Statistical Manual of Mental Disorders, 5th Revision (DSM-5). For example, compositions or pharmaceutical compositions of the present invention containing synapse formation promoters, synapse maintenance agents, neuronal cell function modifiers that cause changes (promotion or inhibition) in the electrophysiological properties of nerve cells (such as the properties of conduction and transmission of stimuli), or cytoskeletal modifiers that promote the maintenance and formation of the neuroskeleton can be used in particular to improve or prevent the symptoms of schizophrenia.

[0156] "Autism spectrum disorder" is a general term for developmental disorders characterized by persistent deficits in social communication and interpersonal interaction from early development, as well as behaviors, interests, or repetitive behaviors. For example, compositions or pharmaceutical compositions of the present invention that include synapse formation promoters, synapse maintenance agents, neuronal cell function modifiers that cause changes (promotion or inhibition) in the electrophysiological properties of nerve cells (such as the properties of conduction and transmission of stimuli), or cytoskeletal modifiers that promote the maintenance and formation of the neuroskeleton can be used in particular to improve or prevent symptoms of autism spectrum disorder. Autism spectrum disorder includes classic autism, Asperger's syndrome, pervasive developmental disorder not otherwise specified (PDD-NOS), Rett syndrome, and childhood disintegrative disorder.

[0157] "Mitochondrial disease" refers to a representative disease caused by mitochondrial dysfunction, and symptoms are often particularly pronounced in the brain, central nervous system, and muscles, which have high energy demands. For example, a composition or pharmaceutical composition of the present invention containing a synapse formation promoter, a synapse maintenance agent, a neuronal cell function modifier that causes changes (promotion or inhibition) in the electrophysiological properties of nerve cells (such as the properties of conduction and transmission of stimuli), or a cytoskeletal modifier that promotes the maintenance and formation of the neuroskeleton can be used in particular to improve or prevent symptoms of mitochondrial disease (such as seizures, stroke-like episodes, and cerebellar ataxia).

[0158] "Dementia" refers to a disease characterized by core symptoms such as memory impairment, disorientation, behavioral disorders, impaired comprehension and judgment, and executive function disorders, which appear as a result of damage to brain cells. For example, compositions or pharmaceutical compositions of the present invention that include synapse formation promoters, synapse maintenance agents, nerve cell function modifiers that cause changes (e.g., acceleration) in the electrophysiological properties of nerve cells (such as the properties of conduction and transmission of stimuli), or cytoskeletal modifiers that promote the maintenance and formation of the neuroskeleton, can be used in particular to improve or prevent the symptoms of dementia. Examples of dementia include Alzheimer's disease, vascular dementia, Lewy body dementia, and frontotemporal dementia, depending on the underlying disease or disorder, but dementia in the present invention includes all of these, and the underlying disease or disorder is not particularly limited.

[0159] "Alzheimer's disease" refers to dementia characterized by atrophy of the medial temporal lobe (hippocampus) as seen in CT and MRI scans, and with event memory impairment as a core symptom of cognitive and memory impairment. It may also be accompanied by other cognitive impairments such as constructional atrophy, visuospatial cognitive impairment such as topographic disorientation, and recent memory impairment. It is a general term for dementia caused by Alzheimer's disease, which is characterized by amyloid-beta aggregation, and dementias with symptoms similar to Alzheimer's disease.

[0160] Vascular dementia refers to dementia that arises in association with cerebrovascular disease and is accompanied by impairments in complex attention, including information processing speed, and frontal lobe executive functions. Depending on the type of vascular disease, it is classified into polyinfarct dementia, small vessel disease dementia, hypoperfusion vascular dementia, hemorrhagic vascular dementia, etc. It frequently coexists with Alzheimer's disease.

[0161] Lewy body dementia is a type of dementia characterized primarily by the appearance of inclusion bodies (Lewy bodies) resulting from the abnormal accumulation of alpha-synuclein in nerve cells, accompanied by symptoms such as cognitive fluctuations, hallucinations, and / or idiopathic parkinsonism. It may also be accompanied by constructional disorders, severe hypersensitivity to antipsychotic drugs, and / or REM sleep behavior disorder. Lewy bodies may also be observed outside the central nervous system.

[0162] Frontotemporal dementia is a type of dementia characterized by brain atrophy in the frontal and / or temporal lobes, accompanied by progressive aphasia. It may also be accompanied by social cognitive impairments such as personality changes and executive function disorders, although memory impairment is often relatively mild. It is broadly classified into conditions involving tau accumulation and conditions involving TDP-43 accumulation, but conditions involving the accumulation of FUS (fused in sarcoma) are also known.

[0163] 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.

[0164] For example, the pharmaceutical composition of the present invention may be a pharmaceutical composition for the treatment of epilepsy, a pharmaceutical composition for the treatment of dementia, a pharmaceutical composition for the treatment of Alzheimer's disease, a pharmaceutical composition for the treatment of amyotrophic lateral sclerosis, etc., containing the conjugate or targeting agent of the present invention.

[0165] 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.

[0166] 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.

[0167] <Method for Targeting Central Nervous System Cells> The present invention relates to a method for targeting central nervous system cells. According to the present invention, a conjugate of 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 synaptic vesicles (anti-SYT2 N-terminal antibody), and a labeled substance and / or a physiologically active substance is brought into contact with a secondary motor neuron, and the conjugate is delivered into the cerebrospinal fluid, thereby causing the antibody to be taken up into the central nervous system cell (particularly within the synaptic vesicles of the central nervous system cell), and targeting of the central nervous system cell (e.g., axon terminal, axon, axonal papilla, cell body, dendrite, etc. of the central nervous system cell). In this way, a conjugate containing an intravesicular domain antibody, including an antibody that binds to the intravesicular domain of synaptotagmin 2, is targeted to the central nervous system cell or its synapse. Therefore, the present invention provides a method for targeting central nervous system cells or synapses, and this method includes bringing the conjugate into contact with a secondary motor neuron. The specific details of each step in this method are in accordance with the description of the method for targeting labeled substances and / or physiologically active substances below.

[0168] <Method for Targeting Labeled Substances and / or Bioactive Substances> The present invention relates to a method for targeting labeled substances and / or bioactive substances, comprising the steps of: contacting a pharmaceutical composition and / or targeting agent (hereinafter often referred to as "targeting agent, etc.") containing the conjugate of the present invention with secondary motor neurons (contact step); delivering the conjugate into cerebrospinal fluid (delivery step); and causing the labeled substance and / or bioactive substance to be taken up into the cells of the central nervous system cells (uptake step).

[0169] (Contact step) The secondary motor neurons in this method may include secondary motor neurons of vertebrates (non-human mammals, humans, and other vertebrates). The secondary motor neurons in this method preferably include human secondary motor neurons.

[0170] This method is typically performed in vivo, and contact in this method is usually achieved by administering a conjugate.

[0171] The organisms to which the present invention is administered are not particularly limited, but for example, the cells may be from the organisms mentioned above (for example, primates such as humans), or they may be mammals other than humans.

[0172] The method of administration is not particularly limited as long as it allows administration into the peripheral blood, 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.

[0173] 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 1 mg / kg or more, 1.5 mg / kg or more, 2 mg / kg or more, 2.5 mg / kg or more, 3 mg / kg or more, 3.5 mg / kg or more, 4 mg / kg or more, 4.5 mg / kg or more, or 5 mg / kg or more per kg of body weight can be applied. Furthermore, for example, the dosage per kg of body weight can be set to 1 g / kg or less, 900 mg / kg or less, 800 mg / kg or less, 750 mg / kg or less, 500 mg / kg or less, 400 mg / kg or less, 300 mg / kg or less, 200 mg / kg or less, 100 mg / kg or less, 75 mg / kg or less, 50 mg / kg or less, 40 mg / kg or less, 30 mg / kg or less, 25 mg / kg or less, 20 mg / kg or less, 15 mg / kg or less, 10 mg / kg or less, 9 mg / kg or less, 7 mg / kg or less, 6 mg / kg or less, 5 mg / kg or less, etc. Specifically, for example, the dosage can be 1 mg / kg to 1 g / kg, 1.5 mg / kg to 900 mg / kg, 1.5 mg / kg to 800 mg / kg, 1.5 mg / kg to 750 mg / kg, 1.5 mg / kg to 500 mg / kg, 2 mg / kg to 400 mg / kg, 2 mg / kg to 300 mg / kg, 2 mg / kg to 200 mg / kg, 2.5 mg / kg to 100 mg / kg, 2.5 mg / kg to 75 mg / kg, 3 mg / kg to 50 mg / kg, 3 mg / kg to 40 mg / kg, 4 mg / kg to 30 mg / kg, 4 mg / kg to 25 mg / kg, 5 mg / kg to 20 mg / kg, 5 mg / kg to 15 mg / kg, 5 mg / kg to 10 mg / kg, 5 mg / kg to 9 mg / kg, 5 mg / kg to 7 mg / kg, 5 mg / kg to 6 mg / kg, etc. The dosage is preferably greater than the dosage used to target motor neurons with intravesicular domain antibodies, including anti-SYT2 N-terminal antibodies. Furthermore, the dosage is preferably below the concentration at which an effective amount of antibody leaks into the cerebrospinal fluid across the blood-brain barrier upon peripheral blood administration of the antibody.

[0174] Multiple types of targeting agents, etc., may be used in this process. In this case, for example, multiple types of targeting agents, etc., can be used together or separately. Also, multiple types of labeling substances and / or physiologically active substances may be used, for example, multiple types of labeling substances and / or physiologically active substances can be used together or separately. Specifically, for example, labeling substances and physiologically active substances may be used in combination.

[0175] The antibody and the labeling substance and / or bioactive substance may be administered separately (in a non-covalent state) if they can form a conjugate with the labeling substance and / or bioactive substance upon contact with secondary motor neurons. In this case, the timing is not particularly limited as long as the labeling substance and / or bioactive substance can bind to the antibody. For example, the labeling substance and / or bioactive substance can be administered before or simultaneously with the antibody. The same or different methods can be used for each administration.

[0176] This process can be performed multiple times. When this process is performed multiple times, the type of targeting agent and the method of administration used may be the same or different each time.

[0177] (Delivery Process) When the targeted agent is brought into contact with secondary motor neurons, the administered conjugate is taken up by the secondary motor neurons, and a portion of it is released from the central region of the secondary motor neurons (cell body or dendrites, etc.) and delivered into the cerebrospinal fluid.

[0178] The method of delivery into the cerebrospinal fluid is not particularly limited, but typically, it can be delivered by allowing a predetermined amount of time to pass after administration.

[0179] In this case, the time is not particularly limited, as long as it is sufficient for the conjugate to be taken up by the secondary motor neuron, to move within the cell, and to be released. The time required for uptake by the secondary motor neuron can be, for example, the time described later in the uptake process. Specific times for this process can be, for example, 50 hours or more, 60 hours or more, 65 hours or more, 70 hours or more, 72 hours or more, 75 hours or more, 79 hours or more, 80 hours or more, 90 hours or more, 100 hours or more, 120 hours or more, 150 hours or more, 168 hours or more, 200 hours or more, 210 hours or more, 220 hours or more, 230 hours or more, or 240 hours or more.

[0180] If necessary, additional administration of a targeting agent or the like may be performed during this process. In this process, the efficiency of uptake of the targeting agent or the like into motor neurons of the present invention can be improved by activating or promoting the activity of motor neurons. Detailed information on how to activate or promote the activity of motor neurons can be carried out in accordance with the information on central nervous system cells described later. Methods for spontaneous activation include, for example, placing motor neurons in an environment in which they can be active for a sufficient amount of time, but environments in which motor neurons can be active are well known in the art.

[0181] (Intake Process) The conjugate delivered into the cerebrospinal fluid comes into contact with central nervous system cells, and if those central nervous system cells express a membrane protein that is an antigen, the conjugate binds to that membrane protein. As a result, the labeled substance and / or physiologically active substance contained in the targeting agent of the present invention are taken up by the central nervous system cells.

[0182] Labeled substances and / or bioactive substances are taken up by central nervous system cells via endocytosis of synaptic vesicles at synapses. Therefore, by activating or promoting the activity of central nervous system cells, the efficiency of taking up the labeled substances and / or bioactive substances of the present invention into central nervous system cells can be improved. Normally, once the labeled substances and / or bioactive substances of the present invention are taken up by synaptic vesicles, they are delivered to the cell body via retrograde transport through the axon.

[0183] The methods for activating central nervous system cells are not particularly limited, but examples include methods for spontaneously activating central nervous system cells for a sufficient period of time, and methods for activating central nervous system cells or promoting synaptic vesicle endocytosis through artificial stimulation.

[0184] In the method of spontaneously activating the central nervous system, there is no particular limit to the duration of activity, but it can be, for example, 1 hour or more, 3 hours or more, 6 hours or more, 12 hours or more, 18 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, 60 hours or more, 72 hours or more, 100 hours or more, 120 hours or more, 150 hours or more, 168 hours or more, 200 hours or more, or 240 hours or more.

[0185] Methods for activating or promoting the activity of central nervous system cells can be carried out by methods that make the subject actively active (for example, methods that stimulate the subject's brain activity) or by methods that promote the activity of central nervous system cells using chemical substances, etc. Examples of compounds that promote the activity of motor nerves 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, which can be administered to the subject. The compounds used for the above chemical stimulation can be administered at pharmaceutically acceptable concentrations or in a manner that is appropriate. The above compounds can be administered to the subject in such a way that the compound stimulation does not exert any biotoxicity, but if biotoxicity occurs, administration of the above compounds to the subject is not required. When the activity of central nervous system cells is promoted, a similar effect can usually be expected in a shorter time compared to methods that induce spontaneous combustion.

[0186] 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.

[0187] 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.

[0188] (Other matters) 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. Also, 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.

[0189] 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.

[0190] 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.

[0191] In this case, the present invention relates to a method for preventing or treating a condition or disease, comprising the steps of: contacting a secondary motor neuron with a targeting agent containing a conjugate of an antibody capable of binding to the intravesicular domain of a membrane protein present in the synaptic vesicles of a motor neuron (administering it into the peripheral blood); delivering the conjugate into the cerebrospinal fluid; and causing the bioactive substance to be taken up into the cells of the central nervous system. This method can prevent or treat various conditions or diseases as exemplified with respect to the pharmaceutical composition.

[0192] 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 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.

[0193] 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.

[0194] 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.

[0195] <Method for Visualizing Central Nervous System Cells> The present invention relates to a method for visualizing central nervous system cells, comprising the steps of: contacting a visualization agent of the present invention, which includes a conjugate of the present invention, with secondary motor neurons; delivering the conjugate into cerebrospinal fluid; causing the labeling substance to be taken up into the cells of the central nervous system cells; and detecting the signal of the labeling substance.

[0196] In this method, the target of administration is as described above for the targeting method of the labeled substance and / or physiologically active substance, and the target may be a human or a mammal other than a human.

[0197] In this method, the steps of contacting the visualization agent of the present invention with secondary motor neurons, delivering the conjugate into the cerebrospinal fluid, and taking up the labeled substance into the cells of central nervous system cells are the same as the contact, delivery, and uptake steps described above for the method of targeting the labeled substance and / or physiologically active substance, except that the visualization agent is used as the targeting agent.

[0198] 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.

[0199] For example, if the labeling substance is a fluorescent molecule or a radiolabeling substance, a signal can be generated in the target by waiting for a sufficient amount of time for the labeling substance to be taken up by the target central nervous system cells, that is, for a sufficient amount of time for the labeling substance to reach the target central nervous system cells and be taken up by them. This time can be appropriately selected in accordance with the time of the delivery step and the uptake step.

[0200] For example, if the labeling substance is a chemiluminescent substance, this can be carried out by waiting for a sufficient amount of time for the labeling substance to be taken up by the target central nervous system cells, and by adding a substrate or other substance used to generate the signal. This step can be carried out simultaneously with or before the signal detection step described later.

[0201] 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.

[0202] If the labeling substance is a fluorescent substance, for example, excitation light containing the excitation wavelength of the labeling substance can be irradiated onto the target central nervous system cells, and the fluorescence wavelength of the labeling substance can be detected using a detector capable of detecting it. 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.

[0203] The detection of a labeled substance's signal includes detecting the presence, location, or quantity of central nervous system cells (e.g., synapses, cell bodies, etc.) in a sample containing central nervous system cells.

[0204] 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.

[0205] 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.

[0206] <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 the central nervous system cells of the target. If the substance is a physiologically active substance, it can be delivered to cells such as central nervous system cells. If the substance is a labeling substance, it can be used to observe the delivery site of the labeling substance (for example, central nervous system cells 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 composition for targeting central nervous system cells, comprising a conjugate of an antibody capable of binding to the intravesicular domain of a membrane protein present in synaptic vesicles of motor neurons, and a labeling substance and / or a physiologically active substance.

[0207] The present invention provides a method for visualizing target central nervous system cells, comprising administering an effective amount of a conjugate of an intravesicular domain antibody, such as an anti-SYT2 N-terminal antibody, and a labeling substance to the target. The present invention also provides a conjugate of the antibody and the labeling substance, or a composition containing the conjugate, for use in this method. The steps and details thereof included in this method are the same as those of the visualization method described above.

[0208] The present invention provides a method for delivering a bioactive substance to target central nervous system cells, 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. The steps and details thereof included in this method are the same as those of the targeting method described above.

[0209] The present invention provides a conjugate of the intravesicular domain antibody and the labeled substance and / or bioactive substance for use in any of the above methods. For example, the present invention relates to 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 a conjugate of the intravesicular domain antibody and the labeled substance and / or bioactive substance for use in a method for targeting a labeled substance and / or bioactive substance to central nervous system cells or their synapses.

[0210] 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.

[0211] Furthermore, the present invention provides for 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.

[0212] 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.

[0213] <Example 1: Delivery of synaptotagmin 2 antibody to mouse brain nerve cells by intravenous injection> We investigated whether synaptotagmin 2 antibody introduced into the body by intravenous injection could be delivered to brain nerve cells.

[0214] The antibody used for introduction was αSYT2-7 (a synaptotagmin 2 antibody with a variable region containing the amino acid sequences of SEQ ID NOs. 215 and 216) as shown in Table 5.

[0215] The antibody solution was prepared by adding antibodies to PBS and mixing them to a final concentration of 1 mg / mL. The antibody solution was administered to wild-type mice by tail vein injection at a dose of 5 mg / kg.

[0216] After 240 hours following administration, the mice were sacrificed, perfused and fixed with a 4% paraformaldehyde solution, and their brains were removed. The removed brains were post-fixed overnight in a 4% paraformaldehyde solution at 4°C.

[0217] The fixed brain was frozen with tissue embedding medium, and 20 μm thick sections were prepared from this frozen tissue block using a cryostat. After blocking the sections, Donkey anti Human IgG(H+L) Cross-Absorbed Secondary Antibody DyLight 650 (invitrogen; catalog number: SA5-10129) was reacted as a secondary antibody, 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% TritonX-100).

[0218] For fluorescence image acquisition, the Alexa 647 signal detection used a maximum excitation wavelength of 650 nm, a maximum detection wavelength of 665 nm, and an actual detection wavelength of 705 nm. The following exposure times and detection thresholds were used for signal detection. A gamma correction value of 1 was used for all settings.

[0219] Exposure time and detection threshold: Alexa 647 exposure time 200ms; detection threshold 140-450.

[0220] The results are shown in Figures 1-6. Figures 1-6 show fluorescence staining images indicating the location of synaptotagmin 2 antibody in mouse brain sections.

[0221] As shown in Figure 6B, a strong signal was observed in facial motor nuclei cells where the cell bodies of secondary motor neurons are located (dashed box in Figure 6B), confirming that the administered antibody was normally taken up by the secondary motor neurons. Furthermore, as shown in Figures 1-6, unexpectedly, many signals indicating the location of the synaptotagmin 2 antibody were observed in other brain regions as well. Moreover, these signals appeared to be stronger than those observed when typical antibodies are administered into peripheral blood.

[0222] Specifically, signals were observed in primary motor cortex cells, supplementary motor cortex cells, somatosensory cortex cells, insular cortex cells, incomplete insular cortex cells, agranuloscular cortex cells, piriform lobe cortex cells (Figure 1), cingulate gyrus cortex cells, caudal interstitial nucleus cells of the medial longitudinal fasciculus, diagonal band cells, medial septal nucleus cells, ventral pallidal nucleus cells (Figure 2), macular cell preoptic nucleus cells, lateral preoptic nucleus cells (Figure 3), hippocampal pyramidal cell layer cells, dentate gyrus granulosa cells, habenula nucleus cells, lateral geniculate nucleus cells, dorsolateral tegmental nucleus cells, dorsolateral nucleus cells, posterolateral nucleus cells, posterior thalamic nucleus cells, medial inferior thalamic nucleus cells, ventromedial hypothalamic nucleus cells (Figure 4), anterior uncinate gyrus cells, parauncinate gyrus cells, uncinate gyrus cells (Figure 5), etc., as shown in Figures 1-5, which depict coronal sections of the cerebral hemispheres. Furthermore, Figure 6, which shows coronal sections of the cerebellum and brainstem, revealed signals in cerebellar granular layer cells, peripheral zone cells, and trigeminal spinal tract nucleus cells. This result suggests that synaptotagmin 2 antibody administered into the peripheral blood was delivered to these central nervous system cells. These cells are known to express synaptotagmin 2.

[0223] The above findings suggest that administering antibodies against membrane proteins present in synaptic vesicles of motor neurons, such as synaptotagmin 2 antibodies, into the peripheral blood can deliver at least a portion of its constant region to central nervous system cells expressing synaptotagmin 2.

[0224] <Example 2: Confirmation of blood clearance of synaptotagmin 2 antibody by intravenous injection> To clarify whether the delivery of synaptotagmin 2 antibody to central nervous system cells by intravenous injection is specific to antibodies against antigens expressed on motor nerve cells, the blood clearance of synaptotagmin 2 antibody was examined.

[0225] The antibodies to be introduced are radioactive isotopes. 89 αSYT2-1 (a synaptotagmin 2 antibody with a variable region containing the amino acid sequences of SEQ ID NOs. 251 and 252) labeled with Zr, as shown in Table 4, and a similarly labeled anti-keyhole limpet hemocyanin antibody (MBL, M194-3) were used as the control antibody.

[0226] The preparation and administration of the antibody solution were carried out in the same manner as in Example 1. Four hours after administration, PET imaging and CT imaging were performed for 10 minutes each under isoflurane anesthesia. The PET imaging and CT imaging were performed using a small-animal PET / CT scanner Si78 (BRUKER). The imaging conditions and image reconstruction conditions of PET / CT are as follows.

[0227] PET imaging conditions: Energy window 30% (357.7 keV to 664.3 keV); Imaging time 600 seconds; Imaging field of view 80×80×150 mm; Resolution 0.25×0.25×0.25 mm; Image size 320×320×600 pixel; Reconstruction algorithm maximum likelihood - expectation maximization method (using Iteration12 software).

[0228] CT imaging conditions: X-ray source filter AL-1.0 mm; Pixel size 200 μm; X-ray tube current 771 μA, voltage 65 kV; Exposure time 45 ms; Imaging mode Static; Continuous rotation; 7 degrees / second.

[0229] From the composite images of the obtained PET images and CT images, coronal section images and sagittal section images were obtained, and the 89 state of Zr accumulation was analyzed by image analysis. The acquisition and analysis of the images were performed using PMOD software (PMOD Technologies).

[0230] The image analysis was performed according to the following procedure. First, as the analysis target space, a VOI (volume of interest) was set in the space filled with blood in the heart whose position was identified from the composite image of the PET image and the CT image. The SUV (standardized uptake value) was calculated from the radioactivity concentration in the VOI, the total radioactivity of the administered antibody, and the body weight of the mouse according to the following calculation formula. SUV is the 89 value standardized with the radioactivity concentration assumed to be 1 when the Zr-labeled antibody is uniformly distributed in the body.

[0231] SUV (g / mL) = Radioactivity concentration in VOI (MBq / mL) / {Total radioactivity (MBq) / Body weight (g)} Each mouse was euthanized 78 to 79 hours after administration by cardiac blood collection under deep anesthesia via isoflurane inhalation (1.0–4.0%), and blood was collected. The radioactivity of the collected blood was measured using a gamma-ray well scintillation analyzer, and the radioactivity distribution was evaluated according to the following procedure. The measured radioactivity (count rate) was corrected for decay based on the time elapsed since administration of each agent to calculate the radioactivity accumulation rate (%ID), and the corrected radioactivity accumulation rate per unit weight of the blood sample (%ID / g) was calculated. The experiment was performed on each individual three times, and statistical analysis was performed using a t-test.

[0232] The results are shown in Figure 7. As shown in Figure 7A, 89 Four hours after administration of the Zr-labeled antibody, the control antibody (labeled "Cont." in Figure 7), which is an antibody against an antigen not present in the mouse body, and the synaptotagmin 2 antibody (labeled "αSYT2-1" in Figure 7) were found to be present in the blood at similar levels. On the other hand, as shown in Figure 7B, at 78 to 79 hours after administration, the amount of synaptotagmin 2 antibody in the blood had decreased to about half that of the control antibody.

[0233] This suggests that when using a synaptotagmin 2 antibody that has the antigen on the cell membrane of secondary motor neurons, the clearance rate from the blood is higher compared to when administering a normal antibody.

[0234] This suggests that the antibody of the present invention is eliminated from the bloodstream by a function specific to motor nerve cells, and that, combined with the observation of a strong signal in Example 1, it may be transported to the central nervous system by this function.

[0235] For example, Cunningham et al (2016) (doi:10.1093 / brain / aww056) suggested that in Guillain-Barré syndrome, autoantibodies against specific antigens widely present on the surface of nerve cells may be taken up by nerve cells and released from the cell body in the central nervous system.

[0236] From the above, it was suggested that administering a large amount of antibody capable of binding to antigens present at the synapses of motor nerve cells into peripheral blood allows delivery to central nervous system cells that would not normally be accessible by peripheral blood administration, through the following mechanism: (1) The antibody binds to the antigen on the synapse of a secondary motor nerve cell with a synaptic terminal located in the periphery; (2) The antibody is taken up by the secondary motor nerve cell by endocytosis; (3) The antibody moves through the interior of the secondary motor nerve cell to the vicinity of the cell body by retrograde transport; (4) The antibody is released into the cerebrospinal fluid from the vicinity of the cell body of a centrally located secondary motor nerve cell; (5) The antibody binds to the antigen at the synapse of a central nervous system cell expressing the antigen; (6) The antibody is taken up by the central nervous system cell by endocytosis.

[0237] <Example 3: Confirmation of the effectiveness of antibody delivery to the central nervous system by intravenous injection> To clarify whether the delivery of antibodies to central nervous system cells by intravenous injection is specific to antibodies against antigens expressed on motor nerve cells, we compared the results when using synaptotagmin 2 antibody and anti-VGAT (vesicular GABA transporter) antibody.

[0238] The experiment was basically conducted in the same manner as in Example 1, except that, in addition to αSYT2-7 (a synaptotagmin 2 antibody with a variable region having the amino acid sequences of SEQ ID NOs. 215 and 216) listed in Table 5, an anti-VGAT antibody (Synaptic Systems, #131 103) was used as the antibody to be introduced, and the gastrocnemius muscle and spinal cord were excised in addition to the brain.

[0239] Additionally, 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), or anticholine acetyltransferase antibody (α-ChAT antibody; catalog number #NBP1-30052; Novus BioLogicals) as additional primary antibodies.

[0240] 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).

[0241] 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.

[0242] Exposure time and detection threshold: Alexa 488 Exposure time 150ms; detection threshold 130–1500; Alexa 594 Exposure time 100ms; detection threshold 100–2500; Alexa 647 Exposure time 100ms; detection threshold 130–1000.

[0243] The results are shown in Figures 8 to 11. Figure 8 shows the results for the neuromuscular junction, Figures 9 and 10 show the results for the spinal cord, and Figure 11 shows the results for the brain. Here, α-BgtX is a marker for acetylcholine receptors on the muscle cell membrane, SYN1 is a marker for the presynaptic region, and α-ChAT is a marker for choline acetyltransferase in motor neurons.

[0244] As shown in Figure 8, when an anti-VGAT antibody was used, no signal originating from the anti-VGAT antibody was observed at the neuromuscular junction (indicated by the arrowhead in Figure 8A), where signals for α-BgtX and SYN1 were observed (Figure 8A). On the other hand, when an anti-SYT2 N-terminal antibody was used, a signal from the anti-SYT2 N-terminal antibody was observed at the neuromuscular junction (indicated by the arrow in Figure 8B) (Figure 8B).

[0245] Furthermore, as shown in Figures 9 and 10, when an anti-VGAT antibody was used as the antibody, no signal originating from the anti-VGAT antibody was observed in the spinal cord and anterior horn of the spinal cord (shown by the dotted line in Figure 9), where α-ChAT signaling is observed (Figures 9A and 10A). 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 in the anterior horn of the spinal cord (Figures 9B and 10B). VGAT has been reported to be expressed in synaptic vesicles of inhibitory neurons, including inhibitory interneurons, which are also present in the anterior horn of the spinal cord (Krishnan VS, et al, Biogerontology. 2018;19(5):385-399. doi:10.1007 / s10522-018-9765-5), and the anti-VGAT antibody used in this example recognizes its intravesical domain as an antigen. However, as mentioned above, no signal originating from the administered anti-VGAT antibody was observed in the spinal cord. This suggests that the administered concentration of this antibody was not high enough to detect a signal from the antibody leaking into the cerebrospinal fluid across the spinal-brain barrier.

[0246] Furthermore, as shown in Figure 11, similar results were observed in the brain. Since VGAT is expressed in synaptic vesicles of inhibitory neurons, VGAT-expressing cells are distributed across a wide brain region, and if anti-VGAT antibodies leak into the cerebrospinal fluid across the spinal-brain barrier, their signal should be observed across a wide brain region. However, no signal originating from anti-VGAT antibodies was observed (Figure 11A), while the signal from anti-SYT2 N-terminal antibody was observed in the telencephalon, including the hippocampus and neocortex (shown by the dotted line in Figure 11B) (Figure 11B).

[0247] Therefore, the anti-SYT2 N-terminal antibody signal observed here is not due to leakage into the cerebrospinal fluid from the spinal-brain barrier caused by high antibody administration concentrations, but rather is suggested to be based on its uptake by motor neurons from synapses in the spinal-brain barrier.

[0248] <Example 4: Relationship between the dosage of synaptotagmin 2 antibody and its delivery to central nervous system cells> The relationship between the dosage of synaptotagmin 2 antibody and whether or not the synaptotagmin 2 antibody is delivered to central nervous system cells was investigated.

[0249] The experiment was basically conducted in the same manner as in Example 1, except that antibody concentrations of 1 mg / kg and 0.1 mg / kg were administered in addition to 5 mg / kg, and the spinal cord was removed in addition to the brain.

[0250] The results are shown in Figure 12. Figure 12A shows the results for the anterior horn of the spinal cord, and Figure 12B shows the results for the hippocampus of the brain.

[0251] In the anterior horn of the spinal cord, a signal of anti-SYT2 N-terminal antibody was observed even at a low dose of 0.1 mg / kg (indicated by the arrow in Figure 12A), confirming that even with low concentrations of the antibody, anti-SYT2 N-terminal antibody is taken up from the neuromuscular junction to the motor neuron synapse and a considerable amount is taken up by secondary motor neurons.

[0252] On the other hand, as shown in Figure 12B, no signal was observed in the hippocampus of the brain at doses of 0.1 mg / kg and 1 mg / kg (Figure 12A), but a clear signal was observed at a dose of 5 mg / kg.

[0253] This suggests that the targeted agent of the present invention can be delivered to the brain by increasing its administered concentration.

[0254] <Example 5: Pharmacological effects in the brain by intravenous injection of synaptotagmin 2 antibody> We confirmed that drug efficacy in central nervous system cells can be achieved by using a conjugate of synaptotagmin 2 antibody and a drug.

[0255] The conjugates of αSYT2-7 (a synaptotagmin 2 antibody having a variable region with amino acid sequences 215 and 216) and nucleic acid molecules shown in Table 5 were used.

[0256] As nucleic acid molecules, we used a control oligonucleotide having a chemically synthesized scrambled sequence and an antisense oligonucleotide targeting the long non-coding RNA malat1 (SEQ ID NO: 256).

[0257] The control oligonucleotide used had the following composition: 5'-T * C m * A * c m tc m gaac m agtA * G * T * -3' (SEQ ID NO: 255) Note that in the sequence, all nucleoside bonds are phosphorothioate bonds, uppercase letters indicate RNA, and lowercase letters indicate DNA. * This indicates that the base is N and the sugar portion is LNA, C m This indicates that the base is 5-methylcytosine.

[0258] The composition of the malat1 antisense oligonucleotide used is as follows: 5'-G * C m * A * ttc m taatagc m A * G * C m * -3' (SEQ ID NO: 257) Note that in the sequence, all nucleoside bonds are phosphorothioate bonds, uppercase letters indicate RNA, and lowercase letters indicate DNA. * This indicates that the base is N and the sugar portion is LNA, C m This indicates that the base is 5-methylcytosine.

[0259] Conjugates were prepared using oYo-Link Azide (AlphaThera) and LED PX2 Photo-Crosslinking Device (AlphaThera) according to the manufacturer's protocol. The conjugates were prepared to have a drug-antibody ratio (DAR) of 2.

[0260] The conjugate solution used was prepared by adding the conjugate to PBS and mixing it until the final concentration was 1 mg / mL.

[0261] The conjugate solution was administered to wild-type mice by tail vein injection at a dose of 5 mg / kg.

[0262] After 240 hours of administration, the mice were sacrificed, their brains were removed, and RNAlater TM (Invitrogen) The brain was left to stand overnight at 4°C in the solution, and then stored at -60°C until use. From the stored brain, the brainstem and subsequent regions were resected, and the hippocampus was also resected to recover the telencephalon region, which contains a large amount of neocortex.

[0263] RNA was extracted from the recovered telencephalon region samples using the RNeasy Lipid Tissue Mini Kit (Qiagen: Cat# 74804) according to the manufacturer's protocol, and cDNA was synthesized by reverse transcription using the QuantAccuracy, RT-RamDA cDNA Synthesis Kit (TOYOBO) according to the manufacturer's protocol.

[0264] Quantitative PCR was performed using TB Green Premix Ex Taq (Tli RNaseH Plus) (Takara Bio) based on the extracted cDNA. The nucleotide sequences of the primer pairs used to measure the expression level of malat1 are shown in SEQ ID NOs. 258 and 259. The expression level of malat1 was corrected to the expression level of the housekeeping gene GAPDH (the nucleotide sequences of the primer pairs used are SEQ ID NOs. 260 and 261), and calculated as a relative value with the average of the value when using a control oligonucleotide set to 1.

[0265] Each group of experiments consisted of 5 individuals, and statistical analysis was performed using Student's t-test.

[0266] The results are shown in Figure 13. As shown in Figure 13, compared to the case where a control oligonucleotide was used (labeled "Cont." in Figure 13), the expression level of malat1 in the telencephalon region, which contains a large amount of neocortex, was significantly reduced to approximately 0.68 when using the malat1 antisense oligonucleotide, and this reduction showed a statistically significant trend.

[0267] This confirms that by using an anti-SYT2 N-terminal antibody, the drug can actually be delivered to the brain and exert its pharmacological effect. All publications, patents, and patent applications cited herein are incorporated herein by direct reference.

Claims

A targeting agent for central nervous system cells expressing the membrane protein, comprising a conjugate of an antibody capable of binding to the intravesicular domain of a membrane protein present in synaptic vesicles of motor neurons and a labeling substance and / or a bioactive substance, which is taken up into secondary motor neurons. 　 The targeting agent according to claim 1, wherein the membrane protein is a human-derived protein. 　 The targeting agent according to claim 1 or 2, wherein the membrane protein comprises any one protein selected from the group consisting of synaptotagmin 2, synaptic vesicle glycoprotein 2A, synaptogyrin 1, synaptogyrin 3, synaptophysin, and synaptotagmin 1. 　 The targeting agent according to any one of claims 1 to 3, wherein the intravesicular domain is a domain containing four or more amino acids. 　 The targeting agent according to claim 3, 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. 　 The targeting agent according to any one of claims 1 to 5, wherein the labeling substance is a fluorescent molecule. 　 A targeting agent according to any one of claims 1 to 6, comprising the labeling substance which is a nerve cell visualization agent. 　 The targeting agent according to any one of claims 1 to 7, wherein the physiologically active substance is one or more selected from the group consisting of synapse formation promoters, synapse maintenance agents, and neuronal cell function modifiers. 　 A targeting agent according to any one of claims 1 to 8, which is taken up into central nervous system cells by endocytosis. 　 A pharmaceutical composition comprising a targeting agent according to any one of claims 1 to 9. 　 A method for targeting the labeling substance and / or bioactive substance to central nervous system cells expressing the membrane protein, A step of contacting a secondary motor neuron with a targeting agent according to any one of claims 1 to 9 and / or the pharmaceutical composition according to claim 10, which includes a conjugate. The process of delivering the conjugate into the cerebrospinal fluid, A targeting method comprising the step of introducing the labeled substance and / or physiologically active substance into the cells of the central nervous system. 　 A method for visualizing central nervous system cells expressing the aforementioned membrane protein, A step of bringing a neuronal cell visualization agent according to claim 7, which includes a conjugate, into contact with a secondary motor neuron, The process of delivering the conjugate into the cerebrospinal fluid, The process of causing the labeling substance to be taken up into the cells of the central nervous system cells of the secondary motor nerve cells, and step of detecting the signal of the labeled substance A method for visualizing central nervous system cells, including [the specified method].

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