Anti-synaptogyrin 3 antibody

Abstract

The present invention addresses the problem of providing an antibody excellent in binding to an intravesicular domain of synaptogyrin 3.　Provided is a synaptogyrin 3 antibody capable of efficiently targeting a desired substance toward a motor neuron synapse.

Description

Anti-synaptogyrin 3 antibody

[0001] The present invention relates to a monoclonal antibody that binds to the intravesicular domain of synaptogyrin 3. The antibody of the present invention can be used as a targeting agent for delivering a physiologically active substance and / or a labeled substance to motor neurons, and as a pharmaceutical composition containing the same.

[0002] The nervous system, which is made up of the central nervous system and peripheral nervous system, regulates various emotions and the functions of muscles, internal organs, etc. Such nervous function can be impaired by nerve damage, disease, aging, etc., which can impair physical and mental health and have a significant impact on social life. Therefore, maintaining and improving nervous function is an extremely important issue, as it is directly linked to maintaining and improving quality of life (QOL).

[0003] The central and peripheral nervous systems are each composed of neurons, and these cells exchange signals with each other through synapses. A synapse is a junction containing a gap formed between the axon terminal of a neuron (presynapse) and the dendrites of another neuron or a cell such as a skeletal muscle or organ (postsynapse). Signals are transmitted when chemicals released from the presynapse bind to receptors present in the postsynapse. Synapse formation is triggered by the interaction of specific membrane proteins expressed in the presynapse and postsynapse.

[0004] In recent years, compounds and peptides capable of maintaining and improving neural function by promoting synapse formation have been developed. Patent Document 1 describes that certain peptides have the effect of promoting dendrite outgrowth and synapse formation in primary cultured cortical neurons (PCN), and that such peptides are used to treat mild cognitive impairment or early dementia. Patent Document 2 describes that C-terminal fragment β (CTFβ), generated by cleavage of amyloid precursor protein (APP) by β-secretase, promotes synapse formation, and that CTFβ is used to treat neurodegenerative diseases.

[0005] Furthermore, Patent Document 3 describes a method for culturing motor neurons having presynaptic regions using microbeads having LRRTM molecules or fusion proteins containing such molecules immobilized on their surfaces. As such, the development of a technology that enables simple in vitro screening of the effects of drugs on motor neuron function is expected to provide a large number of compounds and drug candidates that can act on motor neurons.

[0006] JP 2012-092048 A JP 2008-143867 A WO2021 / 006075

[0007] The present inventors have developed a method for enabling controlled drug delivery to target motor neurons using antibodies capable of binding to membrane proteins present in synaptic vesicles of motor neurons (PCT / JP2023 / 016125). This method has been confirmed to be able to achieve the pharmacological effects of a physiologically active substance linked to the antibody in target motor neurons using antibodies capable of binding to the intravesicular domains of several types of vesicular membrane proteins, including synaptotagmin 2. However, because such a use method had not previously been anticipated, no excellent antibodies capable of binding to membrane proteins that would be effective in this method were known.

[0008] Therefore, an objective of the present invention is to provide an excellent monoclonal antibody capable of binding to a membrane protein present in the synaptic vesicles of motor neurons, and to provide a means for targeting substances to motor neurons and a means for visualizing the targeting site using the same.

[0009] As a result of extensive research to solve the above problems, the inventors succeeded in obtaining an excellent monoclonal antibody that binds to the intravesicular domain of synaptogyrin 3, and discovered that this antibody can be used to efficiently target desired substances to motor neuron synapses, thereby completing the present invention.

[0010] The present invention was made based on these findings and provides the following: [1] An antibody capable of binding to the intravesicular domain of synaptogyrin 3, comprising a heavy chain variable region comprising an HCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 3, an HCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 4, and an HCDR3 consisting of the amino acid sequence shown in SEQ ID NO: 5, and a light chain variable region comprising an LCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 6, an LCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 7, and an LCDR3 consisting of the amino acid sequence shown in SEQ ID NO: 8. [2] The antibody according to [1], selected from any one of the following (a) to (c): (a) an antibody in which the heavy chain variable region consists of an amino acid sequence having 90% or more sequence identity to SEQ ID NO: 1 and the light chain variable region consists of an amino acid sequence having 90% or more sequence identity to SEQ ID NO: 2; (b) an antibody in which the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 1 with one or more amino acid substitutions, deletions, and / or additions, and the light chain variable region consists of the amino acid sequence of SEQ ID NO: 2 with one or more amino acid substitutions, deletions, and / or additions; or (c) an antibody in which the heavy chain variable region consists of the amino acid sequence of SEQ ID NO: 1 and the light chain variable region consists of the amino acid sequence of SEQ ID NO: 2. [3] A conjugate of the antibody according to [1] or [2] with a labeling substance and / or a physiologically active substance. [4] A nucleic acid molecule having a nucleotide sequence encoding the antibody according to [1] or [2]. [5] A cell comprising the nucleic acid molecule according to [4]. [6] A targeting agent for motor neurons, comprising the antibody according to [1] or [2]. [7] The targeting agent according to [6], further comprising a labeling substance and / or a physiologically active substance. [8] The targeting agent according to [7], comprising a conjugate of the antibody with the labeling substance and / or the physiologically active substance. [9] The targeting agent according to [7] or [8], wherein the labeling substance is a fluorescent molecule.

[10] The targeting agent according to any of [7] to [9], comprising the labeling substance, which is a motor neuron visualization agent.

[11] The targeting agent according to any of [7] to

[10] , wherein the physiologically active substance is one or more selected from the group consisting of a synapse formation promoter, a synapse maintenance agent, a muscle strengthening agent, and a nerve cell function modifying agent.

[12] The targeting agent according to any one of [6] to

[11] , which is taken up into cells by endocytosis.

[13] A pharmaceutical composition comprising the targeting agent according to any one of [7] to

[12] .

[14] A method for targeting the labeled substance and / or the physiologically active substance to motor neurons, comprising the steps of contacting motor neurons with the targeting agent according to any one of [7] to

[12] and / or the pharmaceutical composition according to

[13] , and delivering the targeting agent and / or the pharmaceutical composition to the synapse of the motor neurons.

[15] A method for visualizing motor neurons, comprising the steps of contacting motor neurons with a targeting agent that is the motor neuron visualization agent according to

[10] , delivering the targeting agent to the synapse of the motor neurons, and detecting a signal from the labeled substance.

[16] A method for preventing or treating a condition or disease, comprising the steps of contacting motor neurons with the targeting agent according to any one of [7] to

[12] , and delivering the targeting agent to the motor neuron synapse.

[17] The method for prevention or treatment according to

[16] , wherein the condition or disease is a condition or disease exhibiting decreased neurological function.

[18] The method for prevention or treatment according to

[16] or

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

[19] The method for prevention or treatment according to any of

[16] to

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

[20] Use of an antibody capable of binding to the intravesicular domain of synaptogyrin 3 in the manufacture of a medicament comprising the antibody and a physiologically active substance, wherein the antibody comprises a heavy chain variable region comprising HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 3, HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 4, and HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a light chain variable region comprising LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 6, LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 7, and LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 8.

[21] A composition for targeting motor neurons, comprising an antibody capable of binding to the intravesicular domain of synaptogyrin 3, wherein the antibody comprises a heavy chain variable region comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 3, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 4, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a light chain variable region comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 6, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 7, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 8.

[22] An antibody capable of binding to the intravesicular domain of synaptogyrin 3, for use in a method for preventing or treating a condition or disease, wherein the antibody comprises a heavy chain variable region comprising an HCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 3, an HCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 4, and an HCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 5, and a light chain variable region comprising an LCDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 6, an LCDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 7, and an LCDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 8.

[23] An antibody capable of binding to the intravesicular domain of synaptogyrin 3 for use in a method for targeting a labeling substance and / or a physiologically active substance to a motor neuron, the antibody comprising a heavy chain variable region comprising HCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 3, HCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 4, and HCDR3 consisting of the amino acid sequence shown in SEQ ID NO: 5, and a light chain variable region comprising LCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 6, LCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 7, and LCDR3 consisting of the amino acid sequence shown in SEQ ID NO: 8. This specification includes the disclosure of Japanese Patent Application No. 2023-182983, from which the present application claims priority.

[0011] The present invention provides monoclonal antibodies capable of targeting a desired substance to motor neurons. When the substance is a physiologically active substance or a therapeutic agent, conditions and / or diseases caused by abnormalities in motor neurons can be treated. Furthermore, when the substance is a labeling substance, motor neurons can be visualized.

[0012] Figure 1 shows a schematic diagram of the experimental procedure for presynaptic induction and antibody delivery using LRRTM2 beads. Neurospheres were seeded onto a plate, resulting in the extension of axons from the neurospheres. LRRTM2 beads were then seeded onto the neurospheres, resulting in the induction of presynaptic sites from the elongated axons on the LRRTM2 bead surface. A control antibody or an anti-synaptic vesicle membrane protein antibody was then introduced into the plate with presynaptic sites induced, and neurons were activated to investigate whether the anti-synaptic vesicle membrane protein antibody targeted the presynaptic sites and exerted a pharmacological effect. Figure 2 shows fluorescence images of a control antibody group containing a conjugate of a normal rabbit antibody and monomethyl auristatin E (MMAE). The fluorescent signal shown in the figure is derived from βIII tubulin (Tuj1). The black dashed circle in Figure A indicates the location of the neurosphere. Figures B and C show enlarged images of the area enclosed in the white box in Figure A. The scale bar in Panel A represents 1 mm, and in Panels B and C, the scale bar represents 100 μm. Figure 3 shows fluorescence images of the SYT2 antibody group containing anti-synaptotagmin 2 intravesicular domain (N-terminus) polyclonal antibody (polyclonal anti-SYT2 N-terminus antibody) conjugated with MMAE. The fluorescent signal shown in the figure is the signal derived from Tuj1. Panels B and C show enlarged images of the white-framed area in Panel A. The scale bar in Panel A represents 1 mm, and in Panels B and C, the scale bar represents 100 μm. Figure 4 shows graphs quantifying the pharmacological effect of MMAE. The relative axonal volume in the figures is normalized to the result obtained using a control normal rabbit antibody conjugated with MMAE ("Cont. IgG-MMAE" in the figures). In the figure, the dashed line indicates the position where the relative axon volume is 100%, "MMAE" indicates the results for the MMAE-only group, and "α-SYT2 IgG-MMAE" indicates the results for the SYT2 antibody group, where a conjugate of polyclonal anti-SYT2 N-terminal antibody and MMAE was introduced. In the figure, error bars indicate standard error, "*" indicates p<0.05, and "***" indicates p<0.001. Figure 5 shows a graph quantifying the pharmacological effect based on MMAE.In the figure, the relative axon mass is normalized to the result obtained when a control normal rabbit antibody and MMAE conjugate was used ("Cont. IgG-MMAE" in the figure). The dashed line indicates the position where the relative axon mass is 100%, and "α-SYNGR3 IgG-MMAE" indicates the results for the SYNGR3 antibody group transfected with a polyclonal anti-SYNGR3 intravesicular domain antibody and MMAE conjugate. The error bars indicate the standard error. Figure 6 shows a graph quantifying the pharmacological effect of MMAE. The relative axon mass is normalized to the result obtained when a polyclonal anti-SYNGR3 intravesicular domain antibody and MMAE conjugate was used ("Polyclonal Ab" in the figure). The dashed line indicates the position where the relative axon mass is 100%, and "Monoclonal Ab" indicates the results for the SYNGR3 antibody group transfected with a monoclonal antibody of the present invention and MMAE conjugate. In the figure, error bars indicate standard error, and "***" indicates p<0.001. Figure 7 is a graph showing the ELISA quantification of the binding affinity to synaptogyrin 3 when 100 ng / mL of anti-SYNGR3 antibody was used. In the figure, the relative antibody amount is a value normalized to the result when a polyclonal anti-SYNGR3 antibody was used ("Polyclonal Ab" in the figure) as 1.0. In the figure, the dashed line indicates the position where the relative antibody amount is 1.0, and "Monoclonal Ab" indicates the result when a monoclonal antibody of the present invention was used. In the figure, error bars indicate standard deviation, and "***" indicates p<0.0001. Figure 8 is a graph showing the ELISA quantification of the binding affinity to synaptogyrin 3 when 500 ng / mL of anti-SYNGR3 antibody was used ("Polyclonal Ab" in the figure). In the figure, the relative antibody amount is a value normalized to the result when a polyclonal anti-SYNGR3 antibody was used ("Polyclonal Ab" in the figure) as 1.0. In the figure, the dashed line indicates the position where the relative antibody amount is 1.0, and "Monoclonal Ab" indicates the results when the monoclonal antibody of the present invention was used. In the figure, the error bars indicate the standard deviation, and "****" indicates p<0.0001.

[0013] The present invention provides a monoclonal antibody (referred to as "the antibody of the present invention") that is capable of binding to the intravesicular domain of synaptogyrin 3 and can be used to target a substance of interest to motor neurons.

[0014] <Synaptogyrin 3> As used herein, "Synaptogyrin 3 (SYNGR3)" refers to one of the four-transmembrane proteins belonging to the synaptogyrin family. This protein is present in presynaptic vesicles in motor neurons and is thought to be involved in regulated exocytosis, dopamine recycling, and other processes. Synaptogyrin 3 has three cytoplasmic domains and two intravesicular domains separated by four transmembrane domains.

[0015] The intravesicular domain of synaptogyrin 3 is exposed to the lumen of synaptic vesicles. When synaptic vesicles fuse with the plasma membrane in response to an increase in intracellular calcium ion concentration, the lumen of the synaptic vesicle connects to the extracellular space, temporarily exposing the intravesicular domain of synaptogyrin 3 to the extracellular space. The plasma membrane portion containing synaptogyrin 3 is then retrieved into the cell as synaptic vesicle membrane by endocytosis and reused as synaptic vesicles. At this time, the intravesicular domain of synaptogyrin 3 is again exposed to the lumen of the synaptic vesicle.

[0016] Specifically, an exemplary human synaptogyrin 3 is a protein consisting of 229 amino acids, the amino acid sequence of which is set forth in SEQ ID NO: 9. For example, in SEQ ID NO: 9, the positions of each domain are as follows: the first cytoplasmic domain is the region represented by the amino acid sequence of positions 1 to 29; the first transmembrane domain is the region represented by the amino acid sequence of positions 30 to 50; the first intravesicular domain is the region represented by the amino acid sequence of positions 51 to 69; the second transmembrane domain is the region represented by the amino acid sequence of positions 70 to 90; the second cytoplasmic domain is the region represented by the amino acid sequence of positions 91 to 104; the third transmembrane domain is the region represented by the amino acid sequence of positions 105 to 125; the second intravesicular domain is the region represented by the amino acid sequence of positions 126 to 147; the fourth transmembrane domain is the region represented by the amino acid sequence of positions 148 to 168; and the third cytoplasmic domain is the region represented by the amino acid sequence of positions 169 to 229.

[0017] Sequence information of synaptogyrin 3 from mice and other organisms can be easily obtained from publicly known databases such as the NCBI database.

[0018] As used herein, the term "synaptogyrin 3 intravesicular domain" refers to all or a portion of either of the two intravesicular domains of synaptogyrin 3. Examples of the entire intravesicular domain include the first intravesicular domain represented by the amino acid sequence of positions 51 to 69 in SEQ ID NO: 9 (SEQ ID NO: 10) and / or the second intravesicular domain represented by the amino acid sequence of positions 126 to 147 in SEQ ID NO: 11. The second intravesicular domain represented by SEQ ID NO: 11 is preferred. Examples of portions of the intravesicular domain include regions represented by partial sequences of any length in SEQ ID NOs: 10 and / or 11. A preferred portion of the intravesicular domain includes, for example, the amino acid sequence of positions 127 to 144 in SEQ ID NO: 9 (SEQ ID NO: 12).

[0019] As the antigen peptide used to obtain the antibody, a peptide containing additional amino acids in addition to the amino acid sequence of the intravesicular domain or a part thereof can be used, as long as an antibody capable of binding to the intravesicular domain can ultimately be obtained. Examples of antigen peptides that can be used in this manner include peptides having the amino acid sequence shown in SEQ ID NO: 13 or 14.

[0020] <Antibodies of the Present Invention> The antibodies of the present invention are monoclonal antibodies (referred to as "antibodies of the present invention") that can specifically recognize and bind to the intravesicular domain of synaptogyrin 3. The antibodies of the present invention bind to the intravesicular domain of synaptogyrin 3, but preferably do not bind to the intravesicular domain of other membrane proteins belonging to the synaptogyrin family (e.g., synaptogyrin 1 and synaptogyrin 4). Furthermore, the antibodies of the present invention bind to the intravesicular domain of human synaptogyrin 3, but may or may not be able to bind to the intravesicular domain of synaptogyrin 3 derived from other animals (e.g., mouse), for example.

[0021] In this specification, the terms "bind" and "specifically bind" are not particularly limited, but include the following: the binding between an antigen and an antibody is -8 This may mean that the antibody has a binding affinity with a KD value of M or less. The antibody of the present invention has the above-mentioned binding affinity for the intravesicular domain of synaptogyrin 3. It is preferable that the antibody of the present invention does not have the above-mentioned binding affinity for other antigens including other proteins (for example, the intravesicular domain of other membrane proteins belonging to the synaptotagmin family, such as synaptotagmin 1). Alternatively, the binding affinity may be determined by a large binding rate constant. The specific value of the binding rate constant in this case is not particularly limited, but may be, for example, 10 3 / Ms or more, 10 4 / Ms or more, 10 5 / Ms or more (10 x 10 5 / Ms or more, 15 x 10 5 / Ms or more, 16 x 10 5 / Ms or more). Methods for measuring the binding affinity and binding rate constant include, for example, surface plasmon resonance and biolayer interferometry, and for example, biolayer interferometry can be preferably used.

[0022] Alternatively, as used herein, "specifically binds to a target substance" can mean that the binding of a target substance, such as the intravesicular domain of synaptogyrin 3, is at least two-fold, three-fold, or four-fold greater than the binding of a substance other than the target substance, as detected by a typical binding assay using an excess amount of the target substance. Furthermore, when detecting binding by fluorescent labeling as described above, it can mean that the S / N ratio is 2 or greater, 3 or greater, or 4 or greater.

[0023] The antibodies of the present invention may be non-human antibodies, such as those derived from mice, rabbits, or goats, chimeric antibodies, humanized antibodies, or human antibodies. However, when used to treat conditions or diseases in humans, they are preferably, but not limited to, humanized or human antibodies. Therefore, antibodies that can be suitably used in the present invention may be antibodies that contain a framework region (FR) of a human antibody and a complementarity-determining region (CDR) that can confer high binding affinity to the intravesicular domain of synaptogyrin 3.

[0024] The antibody of the present invention may be an IgG antibody molecule or an antigen-binding fragment or derivative thereof. For example, the antibody may be a complete antibody, Fab, Fab', or F(ab')2 fragment, or a single-chain antibody (scFv) fragment in which the heavy chain variable region (VH) and the light chain variable region (VL) are linked via a linker, such as scFv-Fc, sc(Fv)2, Fv, or diabody.

[0025] scFv, scFv-Fc, and sc(Fv)2 are synthetic polypeptides in which variable regions are linked by a linker. The linker may be any linker commonly used in the art, and is not particularly limited. 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.

[0026] The antibodies of the present invention also include derivatives that would be understood by those skilled in the art to the extent that they do not affect antigen-binding ability, such as derivatives modified to facilitate antibody purification or increase stability, and conjugates bound to drugs such as anticancer drugs. As used herein, fragments and derivatives that retain the ability to bind to the intravesicular domain of synaptogyrin 3 are intended to be included in the term "antibody" for convenience, unless the context indicates otherwise.

[0027] The antibodies of the present invention can also be synthesized as multimers, such as dimers, trimers, and tetramers. Furthermore, the antibodies of the present invention may be bispecific antibodies having a first specificity that binds to the intravesicular domain of synaptogyrin 3 and a second specificity that binds to another antigen. The second specificity may be, for example, for another antigen that can be expressed by target cells, or for a physiologically active substance and / or labeling substance to be delivered, and can be selected appropriately depending on the purpose. When the second specificity is for another antigen that can be expressed by target cells, i.e., motor neurons, the use of a bispecific antibody results in more specific delivery to the target cells. Furthermore, when the second specificity is for a physiologically active substance and / or labeling substance, these substances can be delivered to the target site as a complex by administering them together with the bispecific antibody.

[0028] Those skilled in the art can obtain the antibody of the present invention in a form appropriate for the intended use based on the description herein and common general technical knowledge in the art.

[0029] Although not particularly limited, examples of the antibodies of the present invention that bind to the intravesicular domain of synaptogyrin 3 include the following antibodies.

[0030] An antibody comprising a heavy chain variable region comprising HCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 3, HCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 4, and HCDR3 consisting of the amino acid sequence shown in SEQ ID NO: 5, and a light chain variable region comprising LCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 6, LCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 7, and LCDR3 consisting of the amino acid sequence shown in SEQ ID NO: 8.

[0031] In this case, the amino acid sequences of other regions of the present invention (e.g., the amino acid sequences of the FRs and the constant region) are not particularly limited. The binding characteristics of an antibody are basically determined by the CDRs of the heavy and light chains. Therefore, the amino acid sequence of the antibody of the present invention may be modified, but the modification is preferably within the framework region or constant region, from the viewpoint of minimizing the effect on binding characteristics. Furthermore, it is preferable that the modification does not significantly alter the three-dimensional structure of the antibody.

[0032] Furthermore, examples of antibodies that bind to the intravesicular domain of synaptogyrin 3 of the present invention include the following antibodies: (a) An antibody having the above-mentioned HCDR1-3 and LCDR1-3, wherein the heavy chain variable region consists of an amino acid sequence having 90% or more sequence identity to SEQ ID NO: 1, and the light chain variable region consists of an amino acid sequence having 90% or more sequence identity to SEQ ID NO: 2.

[0033] As used herein, "sequence identity" (of amino acid sequences) refers to the percentage (%) of identical amino acid residues in the total number of amino acid residues in the amino acid sequences of two polypeptides being compared, when the sequences are aligned, with gaps inserted appropriately in one or both sequences as needed to maximize the number of identical amino acid residues. Sequence identity can be calculated using techniques and software well known in the art.

[0034] Here, the sequence identity of the amino acid sequence in the antibody of the present invention is not particularly limited, as long as it is 90% or more. For example, the amino acid sequence of the heavy chain variable region of the antibody of the present invention consists of an amino acid sequence that has 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 to SEQ ID NO: 1. Furthermore, the amino acid sequence of the light chain variable region of the antibody of the present invention consists of an amino acid sequence that has 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 to SEQ ID NO: 2, for example.

[0035] Furthermore, examples of antibodies that bind to the intravesicular domain of synaptogyrin 3 of the present invention include the following antibodies: (b) An antibody having the above-mentioned HCDR1 to 3 and LCDR1 to 3, and having a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 1 in which one or several amino acids have been substituted, deleted, and / or added, and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 in which one or several amino acids have been substituted, deleted, and / or added.

[0036] Here, "several" may mean five or fewer, for example, two or more, three or more, four or more, or five. Thus, the antibodies of the present invention may have five or fewer (e.g., two to three) amino acid modifications in each of the heavy chain variable region and / or light chain variable region of the antibody (b) above. Antibodies functionally equivalent to a given antibody can be prepared by techniques well known in the art, such as site-directed mutagenesis.

[0037] As used herein, "amino acid substitution" refers to a substitution among the 20 types of amino acids that constitute natural proteins. The amino acid substitution is not particularly limited, but may be a conservative substitution. A conservative substitution refers to a substitution within a conservative amino acid group that has similar properties, such as charge, side chain, polarity, and aromaticity. Examples of conservative substitutions 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).

[0038] Furthermore, examples of antibodies that bind to the intravesicular domain of synaptogyrin 3 of the present invention include the following antibodies: (c) An antibody whose heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 1 and whose light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 2.

[0039] It is known that after an antibody against a certain antigen is generated in vivo, a process called "affinity maturation" can result in an antibody with improved affinity for the antigen compared to the original antibody. Therefore, based on the amino acid sequences of the specifically disclosed antibodies above, it is possible to obtain antibodies with activities equivalent to or improved over the disclosed antibodies, specifically antibodies with improved binding affinity to the intravesicular domain of synaptogyrin 3 and improved functionality as a targeting agent for substance delivery. These modifications can occur not only in the framework regions and constant regions but also in the CDR regions.

[0040] The method for obtaining the antibody of the present invention is not particularly limited. For example, a non-human mammal can be immunized with the intravesicular domain of synaptogyrin 3, which has been identified as the antigen, and a monoclonal antibody can be obtained by known techniques. The antibody of the present invention can also be obtained by synthesis using genetic engineering techniques 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 nucleotide sequence information of the polynucleotide encoding the antibody.

[0041] When producing an antibody by genetic engineering techniques, polynucleotides encoding the heavy and light chains can be introduced into appropriate host cells, expressed, and the antibody can be obtained as a recombinant protein. In this case, the polynucleotide may be DNA or RNA, and any means used in the art for introduction into the host cell can be used as appropriate. A viral vector, a plasmid vector, a phage vector, or the like can be used as the vector for introducing the polynucleotide into the host cell. Examples of host cells that can be used include bacteria such as Escherichia coli, yeast, insect cells, and animal cells. Here, the polynucleotides encoding the heavy and light chains can be introduced into separate vectors, or can be ligated into the same vector and introduced.

[0042] For example, the antibodies of the present invention, when conjugated with a physiologically active substance, can exhibit excellent physiological activity in motor neurons. The improved physiological activity resulting from the use of the antibodies of the present invention can be confirmed, for example, by administering monomethyl auristatin E (MMAE) and the conjugates of the present invention to motor neurons that have been previously co-cultured with microbeads having LRRTM molecules immobilized on their surfaces and subsequently induced to elongate axons and form presynapses. The targeting effect of the antibodies of the present invention can be confirmed by comparing the axonal mass observed when using a conjugate of MMAE with that observed when using a polyclonal goat anti-SYNGR3 intravesicular domain antibody (Synaptogyrin 3 Rabbit Anti-Human Polyclonal Antibody; Catalog No. LS-C55548-100; antibody from LS Bio, Inc., etc.). For example, the axon mass observed when using the antibodies of the present invention is reduced compared to that observed when using a polyclonal antibody. For example, it is sufficient if there is a statistically significant reduction, and specifically, the average amount of axons can be 95% or less, or 90% or less, of that obtained when a polyclonal antibody is used.

[0043] <Nucleic Acid Molecules> The present invention also provides nucleic acid molecules having a nucleotide sequence encoding an antibody of the present invention. The nucleic acid molecules of the present invention may be any nucleic acid molecule encoding one or more of the antibodies of the present invention. The nucleotide sequence of such nucleic acid molecules is not particularly limited. Examples include codon-optimized nucleotide sequences and nucleotide sequences with an initiation codon (ATG) added to the 5' end. The nucleic acid molecules of the present invention may be DNA, RNA, or a combination thereof, and may contain natural nucleotides as well as modified nucleotides, non-natural nucleotides, etc., as appropriate.

[0044] The nucleic acid molecule of the present invention may contain any other components in addition to the nucleic acid encoding the antibody of the present invention. For example, it may be a gene expression vector that further contains a promoter and is capable of expressing the antibody of the present invention in cells.

[0045] As used herein, the term "gene expression vector" refers to a vector that contains a gene or gene fragment (hereinafter referred to as "gene, etc.") in an expressible state and includes an expression unit that can control the expression of the gene, etc. The gene expression vector may be a plasmid vector, a viral vector, or a phage vector. For example, it may be a plasmid vector that is easy to manipulate for genetic recombination, or a viral vector that can easily introduce genes into immune cells. The vector of the present invention may additionally contain a marker gene (selection marker), an enhancer, a terminator, a replication origin, a polyA signal, etc., as necessary.

[0046] As used herein, the term "expressible state" refers to the state in which a gene to be expressed is placed downstream of a promoter under the control of the promoter.

[0047] The specific type of vector is not particularly limited. For example, the plasmid vector may be a commercially available expression vector for mammalian cells, such as Promega's pCI vector, pSI vector, or pcDNA3 vector, or a shuttle vector that can replicate between mammalian cells and bacteria such as Escherichia coli.

[0048] Viral vectors that can be used include, for example, retroviral vectors (including oncoretroviral vectors, lentiviral vectors, and pseudotyped vectors), adenoviral vectors, adeno-associated virus (AAV) vectors, simian virus vectors, vaccinia virus vectors, Sendai virus vectors, Epstein-Barr virus (EBV) vectors, and HSV vectors. Replication-deficient viral vectors that do not autonomously replicate in infected cells may also be used.

[0049] As used herein, a "promoter" refers to a gene expression regulatory region capable of controlling the expression of a gene or the like located downstream (3'-end side) in a cell into which a gene expression vector has been introduced. Promoters can be classified into ubiquitous promoters (systemic promoters) and site-specific promoters based on the location at which the gene or the like under their expression control is expressed. A ubiquitous promoter is a promoter that controls the expression of a target gene or the like (a target gene or the like) in all cells, i.e., the entire host organism. A site-specific promoter is a promoter that controls the expression of a target gene or the like only in specific cells or tissues. The promoter contained in the gene expression vector of the present invention may be either a ubiquitous promoter or a site-specific promoter, but it is preferable that it can induce expression in host cells.

[0050] Furthermore, promoters are classified into constitutively active promoters, expression-inducible promoters, and stage-specifically active promoters based on the timing of expression. Constitutively active promoters can constitutively express a target gene, etc. in cells. Expression-inducible promoters can induce the expression of a target gene, etc. in cells at any stage. Stage-specifically active promoters can induce the expression of a target gene, etc. in cells only at a specific stage of development. Any of these promoters can be considered as overexpression promoters, as they can cause excessive expression of a target gene in host cells. The promoter contained in the gene expression vector of the present invention is preferably a constitutively active promoter, which enables long-term persistence of therapeutic effects.

[0051] The promoter in the gene expression vector of this embodiment is a promoter that can induce the expression of a nucleic acid encoding an antibody of the present invention in a host cell. Specific examples include a CMV promoter (CMV-IE promoter), an SV40 early promoter, an RSV promoter, an EF1α promoter, a Ub promoter, and a 5' LTR promoter. In the case of a retroviral vector, the nucleic acid encoding the antibody of the present invention can be placed downstream of the 5' LTR promoter to induce its gene expression.

[0052] <Cells> The present invention also provides cells capable of expressing the antibodies of the present invention. The type of host cell of the present invention is not particularly limited. For example, the organism from which the cells are derived is not particularly limited. For example, cells derived from vertebrates, as described below in the section on targeting agents, can be used as the cells of the present invention. The type of host cell is also not particularly limited. For example, the host cell may be an ectodermal cell, a mesodermal cell, an endodermal cell, or a combination thereof. For example, the host cell may be an epithelial tissue, a connective tissue, a cartilage tissue, a bone tissue, a blood tissue (including lymphatic tissue), a muscle tissue, a neural tissue, or a combination thereof. The cell of the present invention may be either a cell without differentiation potential or a cell with differentiation potential (e.g., a multipotent cell, a pluripotent cell, etc.). Specific examples of cells with differentiation potential include mesenchymal stem cells, hematopoietic stem cells, various cancer cell lines, neural stem cells, iPS cells, and ES cells.

[0053] The term "pluripotent stem cells" as used herein refers to cells that have the ability to self-replicate, can be cultured in vitro, and have the pluripotency to differentiate into cells that constitute an individual. Specific examples include embryonic stem cells (ES cells), pluripotent stem cells derived from fetal primordial germ cells (GS cells), and induced pluripotent stem cells (iPS cells) derived from somatic cells. However, human iPS cells or ES cells are preferably used in this method.

[0054] ES cells are often obtained from fertilized eggs, but can also be obtained from sources other than fertilized eggs, such as adipose tissue, placenta, and testicular cells, and all ES cells are within the scope of the present invention. Methods for producing ES cells from sources other than fertilized eggs have been reported (e.g., WO 2003 / 046141), and these reports can be used by referring to them as appropriate.

[0055] iPS cells are artificial stem cells derived from somatic cells. They can be produced by introducing specific reprogramming factors into somatic cells in the form of nucleic acids or proteins. They exhibit properties similar to those of ES cells, such as pluripotency and the ability to proliferate through self-renewal. Examples of genes contained in the reprogramming factors include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, Glis1, or combinations thereof. Many methods for producing iPS cells have been reported, and these reports can be used with appropriate modifications. iPS cells that can be used in this method are preferably human-derived iPS cells, such as human fibroblast-derived iPS cells.

[0056] Many methods for inducing differentiation of human iPS cells into peripheral nerve cells have been reported (e.g., Chambers, Stuart M., et al., Nature biotechnology 27.3 (2009): 275.), and these methods can be used with appropriate modifications by referring to these reports. Alternatively, various nerve cells induced to differentiate from human iPS cells are commercially available and can be purchased from, for example, FUJIFILM Cellular Dynamics, Inc. or ReproCELL Co., Ltd.

[0057] The cells of the present invention are capable of expressing the antibodies of the present invention. Preferably, the cells of the present invention contain the nucleic acid molecules of the present invention. The cells of the present invention can contain multiple copies of the nucleic acid molecules of the present invention.

[0058] <Targeting Agent> The present invention also relates to a targeting agent for motor neurons (referred to as "the targeting agent of the present invention"), which comprises the antibody of the present invention capable of binding to the intravesicular domain of synaptogyrin 3.

[0059] As used herein, the term "targeting agent" refers to an agent for delivering a specific substance to a target. As used herein, a targeting agent is used to transport a labeled substance and / or a physiologically active substance (referred to as a "desired substance") to a motor neuron, particularly to a motor neuron synapse. Furthermore, in targeting synapses and synaptic vesicles, the substance is taken up into the cell, particularly into synaptic vesicles, following endocytosis-mediated recovery of synaptic vesicles, and then delivered to the cell body. Furthermore, the targeting agent of the present invention allows the transported substance to exert its physiological activity in the cytoplasm.

[0060] For example, the targeting agent of the present invention can be incorporated into synaptic vesicles, and the transported desired substance can permeate the synaptic vesicle membrane and migrate into the cytoplasm. Specifically, for example, when a physiologically active substance is used, at least one physiologically active substance in the targeting agent can migrate into the cytoplasm and act on a biological substance in the nucleus, a desired biological substance in the cytoplasm, or a biological substance on the cytoplasmic membrane. The biological substance on which the physiologically active substance acts is not particularly limited as long as it can be present on the cell membrane or inside the cell, and can be, for example, any of polymeric compounds such as proteins and nucleic acids, small molecular compounds such as lipids, sugars, amino acids, and nucleotides, ions such as metal ions, or atoms.

[0061] Specifically, examples of biological substances in the nucleus include DNA, RNA (mRNA, siRNA, miRNA, etc.), transcription factors, nuclear receptors, etc., and examples of biological substances in the cytoplasm include the above-mentioned nucleic acids as well as cytoskeleton, enzymes, metal ions, etc. Examples of biological substances on the cell membrane include lipids in the cell membrane, receptors on the cell membrane, enzymes conjugated with receptors, cell adhesion factors, etc.

[0062] As used herein, the term "motor neurons" refers to a group of neurons that transmit stimuli from the central nervous system to skeletal muscles, which are effector organs. Motor neurons generally include primary motor neurons, which are central neurons, and secondary motor neurons, which are peripheral neurons, but the motor neurons referred to herein are secondary motor neurons.

[0063] As used herein, the term "secondary motor neurons" refers 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. As used herein, motor neurons include, for example, alpha motor neurons, beta motor neurons, and gamma motor neurons. In addition to spinal neurons that have their cell bodies in the anterior nucleus of the spinal cord, the term also includes some cranial nerves, such as the oculomotor nerve, trochlear nerve, abducens nerve, facial nerve, and hypoglossal nerve. Motor neurons as used herein typically secrete acetylcholine as a neurotransmitter and are classified as cholinergic neurons. However, motor neurons that secrete neurotransmitters other than acetylcholine may also be used. The muscle cells to which motor neurons project as used herein are skeletal muscle cells.

[0064] As used herein, "skeletal muscle cells" refers to cells that constitute striated muscles that move the skeleton or cells that have the same phenotype. Skeletal muscle cells, as used herein, broadly encompass muscle cells attached to bones and other muscle cells contained in skeletal muscles, such as muscle spindles. The type of skeletal muscle is not particularly limited, but examples include the diaphragm, vastus lateralis, vastus medialis, rectus femoris, vastus intermedius, biceps brachii, tibialis anterior, tibialis posterior, gastrocnemius, soleus, deltoid, latissimus dorsi, sternocleidomastoid, intercostal muscles, ocular muscles, facial muscles, tongue muscles, and stapedius muscles. Furthermore, skeletal muscle cells, as used herein, also encompass cultured skeletal muscle cells, such as cells differentiated in vitro from artificial stem cells (e.g., iPS cells and ES cells) and / or natural stem cells (e.g., mesenchymal stem cells and skeletal muscle stem cells).

[0065] As used herein, cells may be derived from vertebrates. Vertebrates include fish, reptiles, amphibians, birds, and mammals. Specific examples of mammals include primates (e.g., humans). Cells may also be derived from livestock (chickens, horses, cows, sheep, goats, pigs, etc.), pets (tropical fish, lizards, dogs, cats, rabbits, etc.), or laboratory animals (frogs, mice, rats, monkeys, etc.). Cells do not necessarily have to be derived from a single type of tissue, individual, or animal species; they may be a mixture of multiple types of cells. Furthermore, the health status of the tissue and individual from which the cells are derived is not particularly limited.

[0066] The targeting agents of the present invention can target a desired substance to a motor neuron (e.g., axon terminal, axon, axon hillock, cell body, dendrites, etc. of a motor neuron) via a motor neuron synapse.

[0067] As used herein, "synapse" refers to a junction, including a gap, formed between the axon terminal of a nerve cell and a dendrite of another nerve cell (in the case of the central nervous system) or a cell of a skeletal muscle, organ, etc. (in the case of the peripheral nervous system).

[0068] As used herein, a synapse may be a chemical synapse, such as an excitatory synapse, an inhibitory synapse, etc. As used herein, a synapse may be a synapse formed between a neuron and another neuron (e.g., a synapse formed between an axon of a neuron and a dendrite of another neuron), or a synapse formed between a neuron and a cell of another type (such as a muscle cell), but is preferably a synapse formed by a presynaptic portion of a neuron and a postsynaptic portion on a skeletal muscle cell (also referred to as a "neuromuscular junction").

[0069] As used herein, the term "presynapse" refers to the enlarged portion formed at the axon terminal of a neuron in a synapse, and the term "postsynapse" refers to the portion facing the presynapse in a dendrite of another neuron or in another cell, such as a skeletal muscle or organ. The term "synaptic cleft" refers to the space between the presynapse and the postsynapse. At a synapse, neurotransmitters accumulated in synaptic vesicles present in the presynapse are released into the synaptic cleft and bind to receptors present in the postsynapse, thereby transmitting a signal.

[0070] As used herein, the term "synaptic vesicle" refers to a secretory vesicle present in the cytoplasm of a presynaptic neuron. Synaptic vesicles as used herein include not only vesicles that contain a neurotransmitter and fuse with the cell membrane in response to a stimulus to release the neurotransmitter into the synaptic cleft, but also vesicles that are collected into the neuron by endocytosis (including bulk endocytosis) after neurotransmitter release.

[0071] The targeting agents of the present invention bind to the intravesicular domain of synaptogyrin 3 exposed on the cell membrane in the synaptic cleft, are taken up into synaptic vesicles by endocytosis, and can thereby be delivered to the cell body of a motor neuron, etc. Therefore, the targeting agents of the present invention can target a desired substance (a labeled substance and / or a physiologically active substance) to the interior of a motor neuron, particularly the cell body of a motor neuron. Similarly, for example, when the targeting agents of the present invention are administered into cerebrospinal fluid, the desired substance (a labeled substance and / or a physiologically active substance) can be targeted to the interior of a neuron expressing synaptogyrin 3 on its cell membrane in the central nervous system, particularly the cell body.

[0072] The targeting agent of the present invention may further comprise a desired substance (a labeling substance and / or a physiologically active substance) in addition to the antibody of the present invention capable of binding to the intravesicular domain of synaptogyrin 3.

[0073] As used herein, the term "labeling substance" refers to a substance that emits a signal that allows its presence to be detected. Examples of labeling substances include luminescent labeling substances that emit light under specific conditions, such as fluorescent molecules and chemiluminescent substances, sound-emitting labeling substances that emit sound waves, such as photoacoustic probes, and radioactive labeling substances. Examples of fluorescent molecules include, but are not limited to, 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 Examples of suitable photoacoustic probes include reagents containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 112, 113, 114, 115, 120, 121, 122, 123, 124, 125, 130, 131, 132, 133, 134, 135, 140, 141, 142, 143,

[0074] As used herein, the term "biologically active substance" refers to a substance that can exert a physiological effect directly or indirectly on a living organism or cell. Examples include low-molecular-weight compounds that can exert a physiological effect on target motor neurons, functional medium-sized molecules such as peptides and aptamers, and polymeric compounds including biopolymers such as proteins (e.g., antibodies and enzymes) and nucleic acids (e.g., DNA and RNA). For example, drugs or prodrugs such as synapse formation promoters, synapse maintenance agents, muscle-building agents, or neuronal function modifiers can be used as bioactive substances.

[0075] "Physiological effect" refers to an effect that brings about quantitative and / or qualitative changes in biomolecules such as proteins, DNA, and RNA. As a result of a physiological effect, for example, the function or properties of an organism, organ, tissue, cell, etc. may change. For example, effects such as promotion or inhibition of synapse formation, improvement or prevention of decline in neuronal function, or improvement or prevention of neuronal hyperactivity may be obtained.

[0076] When the targeting agent of the present invention contains a desired substance (a labeling substance and / or a physiologically active substance), the antibody of the present invention and the desired substance are contained in a state where they are not covalently linked (e.g., noncovalently linked) or are covalently linked. When they are contained in a covalently linked state, the antibody of the present invention capable of binding to the intravesicular domain of synaptogyrin 3 and the desired substance form a conjugate (referred to as the "conjugate of the present invention").

[0077] As used herein, the term "conjugate" refers to a substance in which two or more molecules are covalently linked. In particular, the conjugate of the present invention comprises an antibody of the present invention linked to a desired substance (a label and / or a physiologically active substance).

[0078] The covalent and non-covalent bonds between the antibody and the desired substance in the targeting agent of the present invention are not particularly limited, as long as they allow the antibody of the present invention and the desired substance to reach the vicinity of motor neurons in a linked state.

[0079] As used herein, the term "synapse formation promoter" refers to a drug that promotes the formation of presynapses and / or postsynapses. Promotion of synapse formation includes, for example, enhancing synaptic connectivity, (i) increasing the surface area and / or volume of the presynapses and / or postsynapses, (ii) increasing and / or qualitatively altering the amount, density, accumulation rate, accumulation frequency, etc. of proteins specifically expressed in presynapses (e.g., synapsin 1 or synapsin 2), and (iii) increasing and / or qualitatively altering the amount, density, accumulation rate, accumulation frequency, etc. of proteins specifically expressed in postsynapses (e.g., LRRTM family proteins).

[0080] As used herein, the term "synapse maintenance agent" refers to a drug that inhibits or assists in the degeneration of presynapses and / or postsynapses. Synapse maintenance includes, for example, inhibiting or assisting in the weakening of synaptic connectivity, such as (i) inhibiting or assisting in the reduction of the surface area and / or volume of presynapses and / or postsynapses, (ii) inhibiting or assisting in the reduction and / or qualitative change of the amount, density, accumulation rate, accumulation frequency, etc. of proteins specifically expressed in presynapses (e.g., synapsin 1 or synapsin 2), and (iii) inhibiting or assisting in the reduction and / or qualitative change of the amount, density, accumulation rate, accumulation frequency, etc. of proteins specifically expressed in postsynapses (e.g., LRRTM family proteins).

[0081] As used herein, the term "muscle-enhancing agent" refers to a drug that has the effect of enhancing muscle mass or inhibiting muscle weakening, or the effect of promoting such enhancement. The type of muscle strengthening is not particularly limited as long as muscle function is enhanced, but examples include an increase in muscle surface area and / or volume, an increase and / or qualitative change in the number, density, etc. of each element that makes up muscle, such as muscle bundles, muscle fibers, myofibrils, sarcomeres, and muscle cells, and / or an effect of changing the expression level of a specific protein in the cells that make up muscle, an effect of increasing muscle mass and / or muscle strength (e.g., skeletal muscle mass or strength), or an effect of inhibiting muscle weakening through these effects.

[0082] Specific examples of synapse formation promoters, synapse maintenance agents, and muscle strengthening agents include, but are not limited to, compounds discovered by the present inventors and disclosed in Japanese Patent Application Laid-Open No. 2022-053535 (e.g., thiamine and its derivatives), and compounds disclosed in Japanese Patent Application Laid-Open No. 2023-028848 (e.g., atropine, busulfan, chromocarb, procainamide, udenafil, propyphenazone, and derivatives thereof).

[0083] As used herein, the term "neuron function modifying agent" refers to an agent that has the effect of altering or promoting the function exerted by a neuron. The modification of neuron function is not particularly limited as long as it changes the degree and / or properties of the neuron's function, but includes, for example, changes in the electrophysiological properties of the neuron (such as the properties of conduction and transmission of stimuli), changes in gene expression patterns, and changes in morphological properties (such as neurite extension, retraction, and branching, and synapse formation and retraction).

[0084] Specific function-modifying agents include, but are not limited to, cytoskeleton-modifying agents such as AP-1 inhibitors (e.g., compounds disclosed in WO2020 / 196725 discovered by the present inventors), FUS inhibitors, SOD1 inhibitors, TDP-43 inhibitors (e.g., anacardic acid compounds), KIF1A inhibitors, and other microtubule polymerization inhibitors (including auristatin drugs such as monomethyl auristatin E (MMAE), monomethyl auristatin F, and auristatin PE).

[0085] As used herein, the term "cytoskeleton modifying agent" refers to an agent that inhibits and / or promotes one or more selected from the group consisting of cytoskeletal formation, maintenance, degradation, branching, migration, and localization. The cytoskeleton modifying agent herein also includes agents that modify cytoskeletal formation, etc. by acting on molecules other than the cytoskeleton. The cytoskeleton includes microtubules, intermediate filaments, and actin filaments. For example, an agent that inhibits cytoskeletal formation and maintenance, specifically, for example, a microtubule polymerization inhibitor, can be used as a cytoskeleton modifying agent.

[0086] These drugs only need to have an action on nerve cells, and do not necessarily have an action specific to motor neurons.

[0087] In the conjugate of the present invention, the antibody of the present invention and a desired substance (a labeling substance and / or a physiologically active substance) may be directly linked by a covalent bond, or they may be indirectly linked via a linker or the like.

[0088] When the targeting agent of the present invention does not include a conjugate, the desired substance preferably has a moiety capable of binding to the antibody of the present invention. Specifically, for example, a desired substance can be covalently bound to another antibody capable of binding to the antibody of the present invention. In this case, the specific configuration of the "another antibody capable of binding to the antibody of the present invention" is as described in the section "<Antibody of the present invention>" except that the antigen is the antibody of the present invention and may be a polyclonal antibody.

[0089] The binding between the moiety capable of binding to the antibody of the present invention and the desired substance is similar to the binding in the targeting agent or conjugate of the present invention. Thus, the moiety capable of binding to the antibody of the present invention and the desired substance may be linked by a non-covalent bond, a direct covalent bond, or an indirect bond via a linker or the like.

[0090] When the targeting agent of the present invention contains a desired substance, the desired substance can be included in the targeting agent of the present invention in the form of a peptide complex in which the desired substance is non-covalently linked to an antibody of the present invention.

[0091] The site at which a desired substance binds to an antibody of the present invention is not particularly limited, as long as it does not impair the binding of the antibody of the present invention to the intravesicular domain of SYNGR3. Specifically, for example, the desired substance can be bound to a site other than the hypervariable region (HVR) or to the constant region.

[0092] Furthermore, the targeting agent of the present invention may contain multiple desired substances, as long as the functions of each substance are not impaired. In this case, for example, the targeting agent may contain multiple of the same substance, or one or more different substances.

[0093] As the linker usable in the present invention, a linker suitably used in the art can be appropriately used. In this case, the structure and chain length of the linker can be appropriately selected within a range that does not impair the function of the resulting conjugate. The linker may be configured, for example, to be cleavable after transport to the synapse. Alternatively, the linker may be configured, for example, to be not cleavable after transport to the synapse.

[0094] The linker may be any linker commonly used in the art, and is not particularly limited, but a peptide linker consisting of 5 to 25, preferably 10 to 20, amino acid residues, such as a GS linker, can be suitably used. Alternatively, a cleavable linker, such as an acid-labile linker, a photolabile linker, a peptidase-sensitive linker, a dimethyl linker, or a disulfide-containing linker, can be used.

[0095] The antibodies of the present invention contained in the targeting agents of the present invention bind to the intravesicular domain of synaptogyrin 3, which is temporarily exposed on the cell surface upon fusion of synaptic vesicles with the plasma membrane, and can be delivered into cells together with synaptogyrin 3 following endocytosis of synaptic vesicles. Therefore, targeting agents or conjugates are preferably designed to deliver a desired substance to synaptic vesicles of a targeted synapse. Characteristics of compounds that can be delivered to synaptic vesicles are well known in the art. Since the diameter of endosomes in bulk endocytosis is 90 nm to 160 nm, the mean (or median) particle size of the targeting agents or conjugates of the present invention can be, 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. Furthermore, since the diameter of synaptic vesicles is 40 nm to 60 nm, the mean (or median) particle size of the conjugate of the present invention can be, 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. For example, if the targeting agent does not contain the desired substance, the overall particle size when the desired substance is bound to the targeting agent can be within the above-mentioned range. However, for example, the particle size may be designed to be larger for the purpose of inhibiting synaptic vesicle endocytosis or for the purpose of delivering a substance to the synaptic surface or synaptic cleft. Furthermore, for example, if the desired substance is designed to be already separated at the time of delivery to synaptic vesicles, the particle size before separation may be designed to be larger.

[0096] The conjugates or targeting agents of the present invention may be configured to cross the blood-brain barrier, but are not necessarily configured in this way. Typically, the conjugates or targeting agents of the present invention do not cross the blood-brain barrier and therefore do not act on the central nervous system, but only on synapses present in the periphery.

[0097] The synaptic targeting effect on motor neurons can be determined, for example, by administering a conjugate or targeting agent of the present invention containing a physiologically active substance to a subject such as a vertebrate (e.g., a non-human mammal, a human, or another vertebrate) and evaluating the physiological effect of the conjugate or targeting agent of the present invention on the subject's neurons. The physiological effect can be evaluated, for example, by comparing the degree of physiological effect between a group administered with the conjugate or targeting agent of the present invention and a group not administered with the conjugate or targeting agent of the present invention, and / or by comparing the degree of physiological effect between a group administered with the conjugate or targeting agent of the present invention and a group administered with the physiologically active substance alone.

[0098] The present inventors have previously found that presynaptic formation can be induced by co-culturing neurons with microbeads on the surface of which LRRTM molecules (such as the extracellular domain of LRRTM2) are immobilized (WO 2021 / 006075).

[0099] Therefore, whether or not a test substance has a targeting effect on motor neurons containing synapses can also be determined by examining whether or not the test substance is localized at the presynaptic site induced by co-culturing neurons with microbeads.

[0100] "LRRTM (leucine-rich repeat transmembrane neuronal protein) family proteins" refers to proteins belonging to the LRRTM family. The LRRTM family is a family of synaptic organizer proteins on the postsynaptic side and has the activity of inducing 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 the microbeads.

[0101] <Conjugate> The present invention relates to a conjugate of a desired substance (a labeling substance and / or a physiologically active substance) with the antibody of the present invention capable of binding to the intravesicular domain of synaptogyrin 3. The conjugate of the present invention is, for example, taken up into synaptic vesicles of a motor neuron and delivered to the motor neuron.

[0102] <Visualizing Agent for Motor Neurons or Their Synapses> The present invention provides a targeting agent that is an agent for visualizing motor neurons or their synapses (referred to as "the visualizing agent of the present invention").

[0103] The visualization agent of the present invention is a targeting agent containing a labeling substance for use in visualizing motor neurons or their synapses.

[0104] "Visualizing a motor neuron" refers to rendering the entire motor neuron or a portion thereof detectable. When visualizing a portion of a motor neuron, the portion to be visualized may be random or a predetermined portion. For example, a synapse can be visualized as the predetermined portion. In this case, the visualization agent of the present invention can be used as a synapse visualization agent. "Visualizing a synapse" refers to rendering the presynapse and / or postsynapse detectable. Therefore, the visualization agent of the present invention can use any detectable labeling substance in addition to labeling substances that can be detected directly with the naked eye. Similarly, the visualization agent of the present invention can visualize the axon terminal, axon, axon hillock, cell body, dendrites, etc. of a motor neuron.

[0105] The visualization agent of the present invention can be used in vivo or in vitro. Detection of the signal from the labeling substance can be performed while the motor neurons are alive or after the motor neurons are fixed. Labeling substances suitable for detection of live motor neurons are known in the art. Examples include fluorescent substances, luminescent substances such as chemiluminescent or bioluminescent substances, sound-emitting substances such as photoacoustic probes, radioactive substances such as radioisotopes, and contrast agents known in the field of in vivo imaging.

[0106] The visualization agents of the present invention may be used for any application, for example, to visualize the number, size or location of motor neurons or synapses (including neuromuscular junctions) or synaptic vesicles, or to visualize tissue in surgery or diagnosis.

[0107] The visualization agent of the present invention may be provided in the form of a kit together with other reagents, for example, reagents necessary for detecting the labeling substance contained in the visualization agent of the present invention.

[0108] In particular, for example, when the labeling substance is an enzyme or the like, its substrate can be provided together with the visualization agent of the present invention.

[0109] <Composition or Pharmaceutical Composition> The present invention further relates to a composition (referred to as "the composition of the present invention") or pharmaceutical composition (referred to as "the pharmaceutical composition of the present invention") comprising a conjugate or targeting agent of the present invention.

[0110] The composition or pharmaceutical composition of the present invention comprises a targeting agent of the present invention containing a physiologically active substance. In addition to the targeting agent, the composition or pharmaceutical composition of the present invention may optionally contain additives (e.g., 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 depending on the dosage form of the composition or pharmaceutical composition.

[0111] The composition or pharmaceutical composition of the present invention may be prepared in any dosage form, including, but not limited to, a solid formulation, a liquid formulation, a gel formulation, an aerosol formulation, etc. When the composition or pharmaceutical composition is used as a liquid formulation, it can also be prepared as a dry product intended to be reconstituted with, for example, physiological saline immediately before use.

[0112] Examples of excipients include lactose, crystalline cellulose, starch, etc. Examples of binders include starch paste, gum arabic paste, hydroxypropyl cellulose, etc. Examples of disintegrants include starch, celluloses, carbonates, etc. Examples of lubricants include wax, talc, etc.

[0113] When the composition or pharmaceutical composition of the present invention contains a synapse formation promoter, a synapse maintenance agent, a muscle-building agent, or a nerve cell function modifying agent as a physiologically active substance, it can improve or prevent decline in nerve function by promoting synapse formation. Therefore, the composition or pharmaceutical composition of the present invention can be used to improve or prevent decline in nerve function, such as decline in nerve function due to nerve damage, decline in nerve function due to aging, or decline in nerve function due to disease, or to improve nerve function.

[0114] As used herein, "nerve damage" refers to damage at any point on a nerve, and includes damage physically inflicted from outside the body and damage caused by internal factors such as cancer and tumors.

[0115] As used herein, "aging" refers to various functional declines, morphological changes, changes in appearance, and the like that occur in an individual organism over time, as well as the processes involved.

[0116] Frailty and sarcopenia are known conditions caused by aging. Frailty refers to a state in which physical and mental vitality (e.g., motor function, cognitive function) declines with age, and daily living functions are impaired, resulting in physical and mental frailty, due in part to the coexistence of multiple chronic diseases. Examples of declines in physical and mental vitality include cognitive impairment, dizziness, eating disorders, swallowing disorders, visual impairment, depression, anemia, hearing loss, delirium, susceptibility to infection, weight loss, and muscle loss. Examples of chronic diseases include hypertension, heart disease, cerebrovascular disease, diabetes, respiratory disease, and malignant tumors. Meanwhile, sarcopenia refers to a state in which skeletal muscle mass and strength are reduced due to aging or disease. Morphological changes, such as synaptic detachment and partial or complete axonal detachment from postsynaptic sites, have been observed at the neuromuscular junction in aged mice, suggesting that age-related changes in the morphology of the neuromuscular junction are involved in the decline in skeletal muscle mass and other conditions associated with sarcopenia.

[0117] The composition or pharmaceutical composition of the present invention can be used to improve or prevent the decline in neurological function due to aging, particularly in subjects with or at high risk of having frailty or sarcopenia.

[0118] In the present invention, examples of diseases include neurological diseases and neuromuscular diseases. As used herein, the term "neurological disease" refers to a disease caused by damage to nerves such as the central nervous system or peripheral nerves, and includes, for example, one or more diseases selected from the group consisting of Alzheimer's disease, Parkinson's disease, dementia with Lewy bodies, frontotemporal lobar degeneration, progressive supranuclear palsy, corticobasal degeneration, Huntington's disease, dystonia, prion disease, acanthocytic chorea, adrenoleukodystrophy, multiple system atrophy, spinocerebellar degeneration, amyotrophic lateral sclerosis, primary lateral sclerosis, spinal-bulbar muscular atrophy, spinal muscular atrophy, spastic paraplegia, syringomyelia, Charcot-Marie-Tooth disease, frontotemporal dementia, epilepsy, schizophrenia, autism, autism spectrum disorder, and the like.

[0119] As used herein, the term "neuromuscular disease" refers to a disease caused by a disorder of motor nerves, neuromuscular junctions, or muscle cells, and refers to, for example, one or more diseases selected from the group consisting of muscular dystrophy, myopathy, congenital myasthenic syndrome, hereditary periodic paralysis, myasthenia gravis, Lambert-Eaton syndrome, etc.

[0120] 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 the initial pathological state is detachment of motor neurons from skeletal muscles 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 synapse formation or inhibit degeneration between skeletal muscles and motor neurons, can be used, in particular, to improve or prevent the decline in neurological function caused by amyotrophic lateral sclerosis.

[0121] For example, the pharmaceutical composition of the present invention can be a pharmaceutical composition for treating amyotrophic lateral sclerosis, a pharmaceutical composition for treating spinal muscular atrophy, etc., which comprises the conjugate or targeting agent of the present invention.

[0122] The composition or pharmaceutical composition of the present invention containing a muscle-building agent can improve or prevent muscle loss by building muscle. Therefore, the composition or pharmaceutical composition of the present invention containing a muscle-building agent can be used to improve or prevent muscle loss, such as loss of muscle strength after trauma or surgery, loss of muscle strength due to aging, or loss of muscle strength due to disease, or to improve muscle function.

[0123] As used herein, the term "trauma" refers to damage to tissues or organs caused by external factors, and includes, for example, wounds, fractures, sprains, ruptured internal organs, burns, frostbite, and the like.

[0124] The composition or pharmaceutical composition of the present invention containing a function-modifying agent can improve or prevent neuronal decline or hyperactivity by modifying the function of motor neurons. Thus, the composition or pharmaceutical composition of the present invention containing a function-modifying agent can also be used to improve or prevent neuronal hyperactivity, for example, neuronal hyperactivity due to a disease or condition, or to improve or prevent muscle tension, for example, tremors due to aging, or muscle tension due to injury or disease.

[0125] Specifically, in addition to the various diseases and conditions mentioned above, the composition can be used to improve or prevent, for example, abnormal involuntary movements (dyskinesia) (e.g., abnormal head movements, tremors, (painful) convulsions, muscle fasciculations, etc.), abnormalities in walking and mobility (e.g., ataxic gait, difficulty walking, etc.), other coordination disorders (e.g., ataxia, etc.), and other conditions related to the nervous system and musculoskeletal system (e.g., tetany, abnormal reflexes, abnormal posture, spasticity, hypertonia, myotonia, hyperactive deep tendon reflexes, dysphagia, etc.).

[0126] As used herein, "disease" refers to a pathological condition that can be classified by identifiable symptoms or causes in a subject, and includes illnesses and disorders. As used herein, "condition" refers to a pathological condition that includes identifiable symptoms in a subject, but does not fall under the category of a disease.

[0127] The compositions or pharmaceutical compositions of the present invention may be configured to exert a pharmacological effect in any cells other than motor neurons in which synaptogyrin 3 can be expressed. In this case, the specific target diseases and conditions vary depending on the target cells and are not particularly limited. For example, the target diseases and conditions may include any of the diseases and conditions described above, or may include other diseases and / or conditions. Examples of other diseases and / or conditions include, but are not limited to, infectious diseases (viral, bacterial, etc.), cardiovascular diseases, blood diseases, digestive system diseases, immune system diseases (inflammatory diseases, autoimmune diseases, etc.), cancer, lifestyle-related diseases, autonomic nervous system disorders, etc.

[0128] The composition or pharmaceutical composition of the present invention may contain multiple targeting agents of the present invention or 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 agent contained in the composition or pharmaceutical composition. When multiple targeting agents are contained, targeting agents capable of binding to membrane proteins other than synaptogyrin 3 may be included. Examples of such antibodies include anti-synaptotagmin 2 antibodies (anti-synaptotagmin 2 intravesicular domain (N-terminus) antibodies, etc.). Furthermore, for example, targeting agents containing antibodies capable of binding to different sites (e.g., different epitopes or different intravesicular domains) of synaptogyrin 3 may be included. The labeling substances and / or physiologically active substances contained in these targeting agents may be different or the same.

[0129] The nucleic acid molecules and / or cells of the present invention can be comprised in a composition or pharmaceutical composition of the present invention.

[0130] <Method for Targeting Motor Neurons or Synapses> The present invention relates to a method for targeting motor neurons or synapses. According to the present invention, by contacting a motor neuron with an antibody of the present invention that binds to the intravesicular domain of synaptogyrin 3, the antibody can be taken up into the motor neuron (particularly into synaptic vesicles of the motor neuron), thereby targeting a desired substance (a labeling substance and / or a physiologically active substance) to the motor neuron (e.g., the axon terminal, axon, axon hillock, cell body, dendrites, etc. of the motor neuron). In this way, the antibody that binds to the intravesicular domain of synaptogyrin 3 targets the motor neuron or synapse. The desired substance can be delivered into the motor neuron (e.g., into the cell body or synaptic vesicles of the motor neuron) by linking the desired substance to the antibody directly or indirectly via a linker to the antibody, and then contacting the conjugate of the present invention with the motor neuron, or by separately contacting the antibody and a desired substance that can bind to the antibody with the motor neuron. Thus, in accordance with the present invention, there is provided a method for targeting a motor neuron or synapse, the method comprising contacting a cell, such as a motor neuron, with an antibody (optionally linked to a desired substance).

[0131] <Method for targeting labeled substance and / or physiologically active substance> The present invention relates to a method for targeting a desired substance (labeled substance and / or physiologically active substance), which method comprises the steps of contacting a motor neuron with the conjugate or targeting agent of the present invention, and delivering the targeting agent to the motor neuron synapse.

[0132] The target of contact in the present invention is not particularly limited as long as it contains motor neurons. For example, contact may be performed only on motor neurons, or on tissue containing cells other than motor neurons. For example, since the targeting agent of the present invention primarily targets the anterior part of the motor neuron synapse at the neuromuscular junction, it may also be targeted at tissue containing skeletal muscle cells to which motor neurons project.

[0133] The motor neurons used in this method may include motor neurons of a vertebrate (non-human mammal, human, or other vertebrate). Preferably, the motor neurons used in this method include human motor neurons. Hereinafter, the present invention will be described using human motor neurons as an example, but the present method is not limited to human motor neurons.

[0134] Human motor neurons can be used without limitation, regardless of their origin. Examples include, but are not limited to, primary cultures of cells isolated from humans, cells isolated from humans and established as cell lines, and human motor neurons induced to differentiate from human-derived pluripotent stem cells. The pluripotent stem cells from which human motor neurons are derived are preferably human-derived pluripotent stem cells.

[0135] In one embodiment, the method is an in vitro method. In another embodiment, the method is an ex vivo method. In another embodiment, the method is an in vivo method. When the method is an in vivo method, the subject may be a mammal other than a human.

[0136] When the method is an in vitro method, the method may further include a step of inducing synapse formation. As used herein, "inducing synapse formation" refers to causing the formation of a presynapse in the axon of a neuron and / or causing the formation of a postsynapse in the dendrites of another neuron or in cells such as skeletal muscle or an organ. Synapse formation can also be induced by co-culturing a cell intended to form a presynapse with a cell intended to form a postsynapse. Synapse formation can also be induced by other methods, such as co-culturing a motor neuron with beads coated with the extracellular domain of LRRTM2. The step of inducing synapse formation can be performed simultaneously with or before the step of contacting a targeting agent of the present invention with a motor neuron.

[0137] When the method is an in vitro method, the step of contacting the targeting agent of the present invention with motor neurons is carried out by contacting the targeting agent of the present invention with a sample containing motor neurons. The contacting method is not particularly limited as long as the motor neurons in the sample and the targeting agent can come into contact with each other. For example, the targeting agent can be applied by directly sprinkling, spraying, dripping, or painting the sample, by immersing the sample in the targeting agent, or a combination thereof. Furthermore, when the sample is present in another carrier (e.g., a culture medium, etc.), the targeting agent may be applied by sprinkling, spraying, dripping, or painting the carrier.

[0138] The amount to be applied is not particularly limited and can be appropriately determined taking into consideration the number of motor neurons and other conditions. For example, the concentration of the IgG antibody can be 0.01 μg / mL or more, 0.1 μg / mL or more, 0.2 μg / mL or more, 0.5 μg / mL or more, 0.7 μg / mL or more, 0.9 μg / mL or more, 1 μg / mL or more, 2 μg / mL or more, 5 μg / mL or more, 7 μg / mL or more, 9 μg / mL or more, or 10 μg / mL or more.

[0139] When the method is an in vivo method, the step of contacting the targeting agent of the present invention with the test sample is carried out by administering the targeting agent of the present invention to a subject.

[0140] The administration method is not particularly limited, and examples include topical administration, enteral administration, and parenteral administration, specifically administration via the skin, inhalation administration, enema administration, eye instillation, ear instillation, nasal administration, intravaginal administration, administration by enteral 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, and intravitreal administration.

[0141] The dosage is not particularly limited and can be appropriately determined taking into consideration the target animal species and other conditions. For example, when administering an IgG antibody to a mouse, the dosage per kg of body weight can be 0.1 mg / kg or more, 0.5 mg / kg or more, 1 mg / kg or more, 2 mg / kg or more, 4 mg / kg or more, or 5 mg / kg or more.

[0142] The targeting agent used in this step does not have to be of one type. For example, multiple types of targeting agents can be used together or separately. Specifically, for example, a targeting agent containing a conjugate and a targeting agent not containing a desired substance (labeled substance and / or physiologically active substance) can be used. Furthermore, the desired substance used does not have to be of one type; for example, multiple types of labeled substances and / or physiologically active substances can be used together or separately. Specifically, for example, a labeled substance and a physiologically active substance can be used in combination.

[0143] The step of contacting the targeting agent of the present invention with motor neurons can be carried out multiple times. When this step is carried out multiple times, the types of targeting agent and cells used, as well as the application and administration methods, may be the same or different each time.

[0144] When the conjugate or targeting agent of the present invention is contacted with a motor neuron, the targeting agent of the present invention is taken up into the motor neuron via synaptic vesicles. When the targeting agent of the present invention is contacted with a motor neuron, the motor neuron can be activated or its activity can be promoted. By activating or promoting the activity of the motor neuron, the efficiency of uptake of the conjugate or targeting agent of the present invention into the motor neuron can be improved. Usually, when the conjugate or targeting agent of the present invention is taken up into synaptic vesicles, it is delivered directly to the cell body via the axon by retrograde transport.

[0145] The method for activating motor neurons is not particularly limited, but examples include a method of spontaneously activating motor neurons for a sufficient period of time, and a method of activating motor neurons by artificial stimulation or promoting endocytosis of synaptic vesicles.

[0146] Methods for spontaneous activation include, for example, placing motor neurons in an environment that allows them to activate for a sufficient period of time. Environments that allow motor neurons to activate are well known in the art.

[0147] In the method for spontaneously activating motor neurons, the time for which the motor neurons are activated (e.g., contact time with the conjugate or targeting agent of the present invention) is not particularly limited, but may be, for example, 1 hour or more, 3 hours or more, 6 hours or more, 12 hours or more, 18 hours or more, or 24 hours or more when the method is an in vitro method. Furthermore, when the method is an in vivo method, the time may 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.

[0148] Methods for activating or promoting motor neuron activity by artificial stimulation include, for example, placing the motor neuron in an environment in which the motor neuron can be actively activated for a sufficient period of time.

[0149] When the present method is an in vitro method, the method of activating or promoting the activity of motor neurons can involve chemical and / or physical stimulation of the motor neurons. Stimuli for activating motor neurons are well known in the art. Examples of compounds used for chemical stimulation include amiodarone, tetraethylammonium, 4-aminopyridine, barium, potassium ion channel inhibitors such as dendrotoxin, sodium channel agonists such as batrachotoxin, calcium channel agonists such as Bay K8644, high concentrations of potassium ions or neurotransmitters, or combinations thereof. Examples of physical stimuli include temperature changes.

[0150] When chemical stimulation is performed, the amount of compound added is not particularly limited, and can be added at a concentration of, for example, 1 μM or more, 10 μM or more, 50 μM or more, or 100 μM or more.

[0151] The duration of the stimulation is not particularly limited and can be set appropriately taking into consideration the type and intensity of the stimulation, etc. For example, the 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.

[0152] When the present method is an in vivo method, the method of activating or promoting motor neuron activity can be performed by activating a subject (e.g., by inducing exercise in a subject or activating brain activity), or by promoting motor neuron activity using chemicals, etc. Compounds that promote motor neuron activity include compounds used in the above-mentioned chemical stimulation, and can be administered to a subject. The compounds used in the above-mentioned chemical stimulation can be administered at a pharmaceutically acceptable concentration or in a manner. The compounds can be administered to a subject so that the compound stimulation does not cause biotoxicity, but if biotoxicity is manifested, it is not necessary to administer the compounds to the subject. When promoting motor neuron activity, it is usually expected to have the same level of effect in a short period of time as methods that cause spontaneous firing.

[0153] When using a targeting agent that does not contain desired substance, desired substance and targeting agent can be contacted with motor neurons together or separately.When the desired substance and targeting agent are contacted separately, as long as the desired substance can be bound to the targeting agent, the timing is not particularly limited.For example, the desired substance can be contacted with motor neurons before or after the contact of the targeting agent with motor neurons.

[0154] The method for each contact 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.

[0155] This method may further comprise, if necessary, a step of confirming whether targeting is successful or not.When the targeting agent used in this method contains a physiologically active substance, for example, as described above in the section on targeting agents, when a physiological effect is observed, targeting can be judged to be successful.In addition, when the targeting agent used in this method contains a labeling substance, when a signal is detected, targeting can be judged to be successful according to the step of detecting the signal of the labeling substance in the visualization method described below.

[0156] When the pharmaceutical composition of the present invention is used as a targeting agent in the present method, the method can be used as a method for preventing or treating a condition or disease.

[0157] In this case, the method may further include a step of allowing the physiological effect to be fully exerted in the subject. For example, if the physiologically active substance used is a substance that can exert its effect alone, the effect can be exerted by placing the subject in an environment that is sufficiently nutritious for a sufficient period of time for the physiological effect to be exerted. Furthermore, for example, if another substance is required for the physiologically active substance used to exert its effect, the substance can be additionally administered.

[0158] In this case, the present invention relates to a method for preventing or treating a condition or disease, comprising the steps of contacting a motor neuron with a targeting agent comprising the antibody of the present invention and a physiologically active substance, and delivering the targeting agent to the motor neuron synapse. This method can prevent or treat various conditions or diseases exemplified in relation to the pharmaceutical composition. The contacting step of this method preferably comprises administering the targeting agent and / or the pharmaceutical composition to a subject.

[0159] Thus, for example, the method of the present invention is a method for improving or preventing a decline in nerve function, such as a decline in nerve function due to nerve damage, aging, or disease, or for improving nerve function. In one embodiment, the condition or disease is a condition or disease exhibiting a decline in nerve function. In one embodiment, the condition or disease is a neurological disease or neuromuscular disease. In one embodiment, the targeting agent comprises a conjugate of the antibody and the physiologically active substance.

[0160] The duration of this step can be appropriately determined depending on the condition of the subject, the type of physiologically active substance, the dosage, etc. For example, the duration may be determined based on the period required for a physiological effect to be exerted when a physiologically active substance is generally administered, or the physiological effect may be confirmed once or multiple times and continued until the physiological effect is fully exerted.

[0161] <Method for visualizing motor neurons or their synapses> The present invention relates to a method for visualizing motor neurons or synapses, which comprises the steps of contacting a motor neuron with the visualization agent of the present invention, delivering the visualization agent to a motor neuron synapse, and detecting a signal from the labeling substance.

[0162] In this method, the motor neurons used for contact are as described above in the method for targeting a labeling substance and / or a physiologically active substance.

[0163] In one embodiment, the method is an in vitro method. In another embodiment, the method is an ex vivo method. In another embodiment, the method is an in vivo method. When the method is an in vivo method, the subject can be a human or a non-human mammal.

[0164] In this method, the step of contacting a motor neuron with a visualization agent of the present invention and the step of delivering the visualization agent to a motor neuron synapse are the same as the step of contacting a motor neuron with a targeting agent of the present invention and the step of delivering the targeting agent to a motor neuron synapse described in the above-mentioned method for targeting a labeled substance and / or a physiologically active substance, except that a visualization agent is used as a targeting agent.

[0165] This method may further include a step of generating a signal from the labeling substance, if necessary. The method for generating a signal is not particularly limited. The method for generating a signal and whether it is necessary can be determined depending on the type of labeling substance used, etc.

[0166] For example, when the labeling substance is a fluorescent molecule or a radioactive labeling substance, a signal can be generated at the target by waiting a sufficient time for the visualization agent to be delivered to the target motor neuron synapse, i.e., a sufficient time for the visualization agent to reach the target motor neuron synapse and for sufficient endocytosis of synaptic vesicles to occur at the motor neuron synapse. This time can be appropriately selected based on the time required for the step of delivering the visualization agent to the motor neuron synapse.

[0167] For example, when the labeling substance is a chemiluminescent substance, this can be achieved by waiting a sufficient time for the visualization agent to be delivered to the target motor neuron synapse and then adding a substance such as its substrate used to generate a signal. This step can be carried out simultaneously with or before the step of detecting a signal, which will be described later.

[0168] This method further comprises the step of detecting the signal of the detection substance. The method used for detection is not particularly limited and can be appropriately selected depending on conditions such as the type of labeling substance used.

[0169] When the labeling substance is a fluorescent substance, for example, excitation light containing light of the excitation wavelength of the labeling substance is irradiated onto the motor neuron, and the fluorescence wavelength of the labeling substance can be detected using a detector capable of detecting the fluorescence wavelength of the labeling substance. When the labeling substance is a chemiluminescent substance, for example, detection can be performed using a detector capable of detecting the emission wavelength of the labeling substance. When the labeling substance is a radioactive labeling substance, detection can be performed using a detector capable of detecting the radiation emitted by the labeling substance.

[0170] Detection of the signal of the labeling substance includes detecting the presence, location, or quantity of motor neurons (eg, synapses, cell bodies, etc.) in a sample containing motor neurons.

[0171] In addition to detecting the signal of the labeled substance in the sample, the method of the present invention may further include a step of determining the presence, location, or amount of the signal of the labeled substance detected in the sample by comparing it with the signal in a standard sample containing the labeled substance or with a previously prepared reference value. The standard sample is not particularly limited as long as it is a biological sample that serves as a reference for determining whether or not a specific condition or disease exists. Specific examples include those obtained from healthy individuals, those obtained from the same individual as the sample at a different collection time, and those obtained from an individual known to have a specific condition or disease. The standard sample may be, for example, a biological sample derived from the same biological species, individual, tissue, or cell as the sample, or a biological sample derived from a different biological species, individual, tissue, or cell. Furthermore, the reference value is not particularly limited as long as it serves as a reference for determining whether or not a target condition exists. The reference value can be set, for example, based on the intensity or number of signals typically detected in the standard sample.

[0172] In either case, the method of comparison is not particularly limited, and may be performed, for example, by visual inspection, by the magnitude of the numerical value, or by a statistical method.

[0173] <Other Inventions> The present invention provides a method for administering a desired substance to a subject. The substance is in the form of a conjugate between the antibody of the present invention and the desired substance. This allows the desired substance to be delivered to cells of the subject, such as motor neurons. If the desired substance is a physiologically active substance, the physiologically active substance can be delivered to cells such as motor neurons. Furthermore, if the desired substance is a labeled substance, the method can be used to observe the delivery site of the labeled substance (e.g., a motor neuron or its synapse). The present invention also provides a conjugate of the antibody and the desired substance for use in this method, or a composition comprising the conjugate. In this case, the present invention relates to a composition for targeting motor neurons or their synapses, comprising the antibody of the present invention or a conjugate of the antibody and a desired substance (a labeled substance and / or a physiologically active substance).

[0174] The present invention provides a method for visualizing motor neurons in a subject, the method comprising administering to the subject an effective amount of a conjugate of the antibody of the present invention and a labeling substance. The present invention also provides a conjugate of the antibody and the labeling substance, or a composition comprising the conjugate, for use in the method.

[0175] The present invention provides a method for delivering a physiologically active substance to motor neurons in a subject, the method comprising administering to the subject an effective amount of a conjugate of the antibody of the present invention and the physiologically active substance. The present invention also provides a conjugate of the antibody and the physiologically active substance, or a composition comprising the conjugate, for use in the method.

[0176] The present invention provides the antibody of the present invention or the conjugate of the present invention for use in any of the above methods. For example, the present invention relates to the antibody of the present invention or the conjugate of the present invention for use in a method for preventing or treating a condition or disease. Furthermore, for example, the present invention relates to the antibody of the present invention or the conjugate of the present invention for use in a method for targeting a labeling substance and / or a physiologically active substance to motor neurons or their synapses.

[0177] The present invention provides the antibody or a conjugate of the antibody and the substance, for use in the manufacture of a medicament for use in any of the above methods.

[0178] The present invention also provides use of the antibody of the present invention or the conjugate of the present invention in the production of a medicament comprising the antibody and a physiologically active substance.

[0179] The present invention will be described in more detail below using examples, although the technical scope of the present invention is not limited to these examples.

[0180] Example 1: Production of monoclonal antibodies Production of monoclonal antibodies and screening by B cell cloning method were outsourced to Medical & Biological Laboratories Co., Ltd. The outline of the production and screening procedures is as follows.

[0181] 1. Synthesis of Antigen Peptides The following peptides (containing partial sequences of the second intravesicular domain) were synthesized as immunization antigens and screening antigens by outsourcing to Eurofins Genomics K.K.

[0182]

[0183] The immunizing antigen was conjugated to a carrier protein, keyhole limpet hemocyanin (KLH), using Ellman's reagent to induce an immune response.

[0184] 2. Immunization of mice: A 1.0 mg / mL solution of KLH-conjugated immunization antigen was prepared, and four mice were immunized once a week for a total of four times. Serum for antibody titer determination and lymph node cells for B cell isolation were collected from the immunized mice.

[0185] The antibody titer of the obtained serum was evaluated by ELISA using the screening antigen peptide synthesized in "1. Synthesis of antigen peptide" as an antigen. As a result, it was confirmed that antibodies capable of binding to the intravesicular domain of human synaptogyrin 3 were present in the collected mouse serum.

[0186] 3. Isolation of B cells Mouse lymph node cells collected for B cell isolation and stored at -80°C were awake and incubated at 37°C in 5% CO2 for 1 hour.

[0187] The cells were collected and stained with an anti-B cell marker antibody and streptavidin (capable of binding to biotin on the screening peptide).

[0188] The stained cells were analyzed using FACS Melody (BD Japan), and cells positive for anti-B cell marker and biotin were isolated individually as B cells reactive to the antigen.

[0189] 4. cDNA synthesis and isolation of variable region sequences mRNA was extracted from each isolated B cell, and cDNA was synthesized by the 5' RACE method.

[0190] Furthermore, nucleic acid fragments containing the variable region sequences were amplified by PCR from the cDNA synthesized from each cell, and agarose electrophoresis confirmed that genes of the desired size (VH, VL) had been amplified.

[0191] 5. Construction of antibody expression vectors Artificial antibody genes were constructed based on the designed gene sequences of the mouse IgG1 constant region (CH) and mouse Igκ constant region (CL) and the variable region genes (VH, VL) amplified in "4. cDNA synthesis and isolation of variable region sequences." Construction of these artificial antibody genes was outsourced to Integrated DNA Technologies, Inc. The amplified VH was inserted into the pcDNA3.4 plasmid together with the human IgG1 constant region (hCH) (heavy chain expression vector), and similarly, the VL was inserted into the pcDNA3.4 plasmid together with the human Igκ constant region (hCL) (light chain expression vector) to construct antibody expression vectors.

[0192] 6. Preparation of antibody-containing supernatants Heavy chain expression vectors and light chain expression vectors were transfected into HEK293T cells, and the culture supernatant was collected three days after transfection as antibody-containing supernatants. Lipofectamine was used for vector transfection. TM 2000 Transfection Reagent (Cat. No.: #11668027, Thermo Fisher Scientific) was used, and Opti-MEM was used as the transfection medium. TM Reduced Serum Medium (Cat. No.: #31985070, Thermo Fisher Scientific) was used. As a negative control, culture supernatant of untransfected HEK293T cells was collected.

[0193] 7. Antibody screening The obtained antibodies were screened by ELISA and flow cytometry. The outline of each screening procedure is as follows.

[0194] 7-1. Screening by ELISA Method Antigen-binding ELISA plates were prepared by adding the screening peptides synthesized in "1. Synthesis of Antigen Peptides" to streptavidin-immobilized Immobilizer Streptavidin plates (catalog number: #436020, Thermo Fisher Scientific). Furthermore, antibody-containing supernatant was added to this antigen-binding ELISA plate as the primary antibody and incubated. A negative control was used in place of the antibody-containing supernatant as the primary antibody. After incubation, the anti-mouse antibody Anti-IgG (H+L) pAb-PE (catalog number: #330, MBL) was added as the secondary antibody and incubated.

[0195] After the secondary antibody reaction, TMB-US (catalog number: #TMB-US, Moss) was added to the plate as a color-developing substrate to develop the label, and the color reaction was stopped using H2PO4 (catalog number: 167-02166, Fujifilm Wako Pure Chemical Industries).

[0196] 7-2. Screening by flow cytometry First, we prepared cells for screening that express the intravesicular domain of human SYNGR3 on the cell surface. HEK293T cells were plated in a 6-well plate (catalog number: #3820, IWAKI) at 5.0 × 10 5 The cells were seeded at a seeding density of 1000 cells / well and transfected with an antigen expression vector. The antigen expression vector used was a plasmid vector prepared by inserting a DNA fragment (SEQ ID NO: 15) containing the gene sequence (SEQ ID NO: 16) encoding human SYNGR3 (amino acid sequence represented by SEQ ID NO: 9) into the multicloning site of the pCMV6-AC-Myc-DDK-IRES-GFP-Puro Mammalian Expression Vector plasmid (ORIGENE). The gene transfection reagent and medium used were the same as those used in "6. Preparation of antibody-containing supernatant."

[0197] Three days after gene transfer, cells were collected for screening and subjected to flow cytometry using a CytoFLEX (Beckman Coulter).

[0198] Cell labeling was performed as follows. The prepared antibody-containing supernatant was added to each screening cell. Anti-Mouse IgG (H+L) pAb-PE (catalog number: #IM0855, Beckman Coulter) was used as the secondary antibody at a 1:200 dilution. For the negative control, negative control supernatant was added as the primary antibody, and the same secondary antibody as that used for the antibody-containing supernatant was added.

[0199] 8. Determination of antibody gene sequence One clone of antibody that showed a reaction in both ELISA and flow cytometry screening was isolated as the monoclonal antibody of the present invention, and the heavy chain expression vector and light chain expression vector used to express that antibody were isolated as a set of antibody expression vectors of the present invention. To identify the sequence of the antibody of the present invention, the nucleotide sequence of the antibody expression vector of the present invention was determined. Sequencing was outsourced to Azenta Inc. and performed by Sanger sequencing.

[0200] The amino acid sequences of the variable regions of the monoclonal antibodies of the present invention are shown in Table 2.

[0201]

[0202] 9. Identification of Complementarity-Determining Region (CDR) Sequences The amino acid sequences of the CDRs of the monoclonal antibodies of the present invention were identified based on the Kabat method. The amino acid sequences of each identified CDR are shown in Table 3.

[0203]

[0204] 10. Preparation of purified antibody solution A set of antibody expression vectors of the present invention was introduced into Expi CHO TM The cells were transfected and the culture supernatant was collected.

[0205] Pierce the obtained culture supernatant TMAntibodies were isolated using 10 mL disposable columns (catalog number: #29924; Thermo Scientific). Elution with 0.1 M citrate buffer (pH = 4.0) was repeated until the recovered antibody concentration reached 0.1 mg / mL or less, and fractions with antibody concentrations of 0.1 mg / mL or higher were isolated as the antibody solution. The antibody solution was dialyzed against 1x Dulbecco's phosphate-buffered saline (catalog number: D1408; Sigma-Aldrich) and filtered through a 0.22 μm pore size filter to prepare a purified antibody solution.

[0206] Example 2: Evaluation of monoclonal antibodies The antigen-binding properties of the obtained monoclonal antibodies were evaluated.

[0207] Using sc-hSYNGR3 as an antigen, the binding properties of the antibodies were measured using the Octet RED96e System (SARTORIUS). The measurement conditions were as follows: Biosensor: Streptavidin biosensor (catalog number: #18-5019; SARTORIUS); Antibody concentrations: 10 nM / 5 nM / 2.5 nM / 1.25 nM / 0.63 nM / 0.31 nM / 0.17 nM / 0 nM; Buffer: PBS containing 0.02% Tween-20 and 0.01% BSA; Equilibration before immobilization: Incubate with buffer only for 60 seconds; Immobilization: Incubate with buffer containing antigen peptide for 300 seconds; Equilibration before antibody addition: Incubate with buffer only for 300 seconds; Antibody binding reaction: Incubate with buffer containing antibody for 300 seconds; Antibody dissociation reaction: Incubate with buffer only for 900 seconds; All incubations were performed at 30°C and agitation speed of 1000 rpm.

[0208] As a control, a polyclonal goat anti-SYNGR3 intravesicular domain antibody was used, and the antigen was used at a concentration of 0.02 μg / mL.

[0209] The measurement results are shown in Tables 4 and 5. Table 4 shows the results for the polyclonal goat anti-SYNGR3 intravesicular domain antibody, and Table 5 shows the results for the monoclonal antibody of the present invention.

[0210]

[0211]

[0212] The binding rate constant is a value that indicates how easily the binding reaction proceeds, and the higher the affinity, the larger the value.

[0213] The results shown in Tables 4 and 5 indicate that the monoclonal antibodies of the present invention have a higher binding rate constant and higher affinity with the antigen than the results measured with the control polyclonal antibody.

[0214] Example 3: Verification of pharmacological effects of drug delivery using intravesicular domain antibodies Using synaptotagmin 2 (SYT2) as an example of a vesicular membrane protein, we investigated whether drug-based pharmacological effects would be observed when drugs were delivered into motor neurons using antibodies that bind to its intravesicular domain (N-terminus).

[0215] 1. Cell culture Human iPS-derived motor neurons (40HU-005-2M; iXCells Biotechnologies) were thawed using a Dead Cell Removal Kit (Veritas) according to the kit's protocol. The thawed cells were plated at 2 × 10 4 Neurospheres were generated by culturing the cells at a density of 100 cells / well in Motor Neuron Maintenance Medium (iXCells Biotechnologies) for 1 week. The neurospheres were selected based on size and circularity, and those that met these criteria were used in the following experiments.

[0216] After coating 96-well EZVIEW® Culture Plates LB (AGC Technoglass Corporation) with poly-D-lysine and Geltrex® Matrix (Thermo Fisher Scientific), the selected neurospheres were seeded onto the plates and cultured for 20 days. The motor neuron culture medium described above was used as the initial medium, and on the second day of culture, the medium was replaced with the same medium. Thereafter, the medium was replaced three times weekly with neuronal medium (Neurobasal plus medium (supplemented with B27 plus supplement (Thermo Fisher Scientific), 20 ng / mL BDNF, 20 ng / mL GDNF, and penicillin-streptomycin)). Each medium change was performed at 50 μL per well.

[0217] 2. Preparation of LRRTM2 beads Microbeads coated with the extracellular domain of LRRTM2 (active beads) were prepared as disclosed in WO2021 / 006075.

[0218] Specifically, streptavidin-coated microspheres (Streptavidin Coated Microspheres; Bangs Laboratories, Inc.; polystyrene, average diameter 9.94 μm) were washed twice with wash buffer (phosphate-buffered saline (PBS), 0.01% bovine serum albumin (BSA), 0.05% Triton X-100) and reacted with biotinylated anti-human IgG (Fc-specific) antibody (Sigma-Aldrich; mouse monoclonal) in binding buffer (PBS, 0.01% BSA) to immobilize the biotinylated anti-human IgG (Fc-specific) antibody on the streptavidin-coated microbeads. The resulting beads were washed three times with wash buffer (anti-human IgG Fc antibody beads).

[0219] Next, the anti-human IgGFc antibody beads were suspended in binding buffer, and a fusion protein of the extracellular domain of human LRRTM2 and the Fc portion of human IgG (LRRTM2-Fc; R&D Systems) was added to immobilize LRRTM2-Fc on the anti-human IgGFc antibody beads. The resulting beads were washed with washing buffer and suspended in binding buffer (LRRTM2 bead suspension).

[0220] 3. Induction of presynaptic sites (Figure 1) After 20 days of culture, LRRTM2 beads were seeded at 0.1 μg / well on plates and cultured at 37°C for 48 hours to induce the formation of presynaptic sites.

[0221] 4. Preparation of conjugates The drug used was monomethyl auristatin E (MMAE: MedChemExpress), a microtubule polymerization inhibitor. MMAE-antibody conjugates were prepared using MagicLink TM The conjugate was prepared using a kit (BroadPharm) according to the manufacturer's protocol.

[0222] The antibodies used were polyclonal rabbit anti-SYT2 N-terminal antibody (Polyclonal rabbit purified antibody SYT2 lumenal domain; catalog number 105 223; Synaptic Systems) or, as a control, normal rabbit IgG antibody (Normal Rabbit IgG; catalog number AB-105-C; R&D Systems).

[0223] 5. Preparation of Conjugate Solution: Neuronal culture medium was warmed at 37°C for 30 minutes, and then 4-aminopyridine (Sigma-Aldrich) was added to the medium to a final concentration of 100 μM. The conjugate was then added to a final concentration of 1 μg / mL and mixed. The crude conjugate solution was centrifuged at 200 g for 3 minutes at room temperature, and the supernatant was collected as the conjugate solution.

[0224] As an additional control, 13 samples were loaded with MMAE alone rather than the conjugate, and the MMAE solution was prepared as described above, using the same concentration of MMAE instead of the conjugate.

[0225] 6. Conjugate Introduction In the plate on which the presynapse had been formed, the medium was replaced with 100 μL of the conjugate solution or MMAE solution, and the plate was cultured at 37° C. for 30 minutes to introduce the conjugate.

[0226] After transfection, the solution was collected and washed, and the medium was replaced with neuronal medium containing neither the conjugate nor MMAE. After that, the cells were cultured for an additional 24 hours to allow axonal outgrowth.

[0227] 8. Fixation of Cells After culturing, the cells were washed with neuronal medium and then fixed with 2% paraformaldehyde (PFA) solution.

[0228] 9. Immunocytochemical Staining Immunocytochemical staining was performed using the added antibody as the primary antibody. After fixed cells were permeabilized with detergent and blocked, primary antibody reactions were performed using mouse anti-βIII tubulin (Tuj1) antibody (catalog no. 801202; Biolegend) as an additional primary antibody. Subsequently, secondary antibody reactions were performed using Alexa 555-conjugated anti-mouse antibody (catalog no. A32727; Thermo Fisher Scientific), and fluorescent images were acquired. Blocking was performed using blocking buffer (PBS + 2% normal goat serum + 1% BSA + 1% fetal bovine serum + 0.02% Triton X-100). The number of samples was as follows: 13 samples in the MMAE-only group; 17 samples in the control antibody group; and 27 samples in the polyclonal SYT2 antibody group.

[0229] Fluorescence images were acquired using an inverted live cell (DMi8) fluorescence microscope (Leica) equipped with LAS X software (Leica) at the following excitation wavelength, detection wavelength, exposure time, and detection threshold. A gamma correction value of 1 was used for all images.

[0230] Excitation and detection wavelengths: Alexa 555 maximum excitation 555nm; maximum detection 580nm; actual detection 595nm. Exposure time and detection threshold: Alexa 555 exposure time 50ms; detection threshold 100-3500.

[0231] 10. Analysis of axonal volume Axonal volume was calculated as the total brightness of the Tuj1 signal in the acquired fluorescent images (= (fluorescence intensity per pixel) × (number of pixels)). The result obtained using a control normal rabbit antibody conjugate with MMAE was set at 100%, and the normalized value was used to calculate the relative axonal volume. Brightness was acquired using LAS X software (Leica).

[0232] 11. Statistical Analysis Statistical analysis was performed using GraphPad Prism 9 (GraphPad Software) by t-test, with p<0.05 used as the significance level.

[0233] The results are shown in Figures 2 to 4. Figures 2 and 3 show immunocytochemical staining images of axons in the control antibody group (Figure 2) using a control normal rabbit antibody conjugated with MMAE, and the SYT2 antibody group (Figure 3) using a polyclonal anti-SYT2 N-terminal antibody conjugated with SYT2. Figure 4 is a graph quantitatively showing the results.

[0234] Under all experimental conditions, axons extended from the neurospheres, and presynaptic sites were also guided normally (Figs. 2A and 3A).

[0235] MMAE inhibits the polymerization of microtubules, a cytoskeleton important for axonal elongation and maintenance, so the stronger the effect of MMAE, the more axonal elongation and maintenance are inhibited, leading to a decrease in axon mass.

[0236] In the control antibody group, many thick axons and presynaptic bulges were observed even in the distal region far from the neurosphere (Fig. 2B), while in the proximal region relatively close to the neurosphere, axons were observed running in a nearly parallel array (Fig. 2C).

[0237] In contrast, in the SYT2 antibody group, only a few thin axons were observed in the distal region far from the neurosphere, and presynaptic formation was poor (Fig. 3B).Furthermore, in the proximal region relatively close to the neurosphere, axonal trajectories were not as well aligned as in the control antibody group, and axonal trajectories appeared disorganized (Fig. 3C).

[0238] Quantitative analysis confirmed these observations. Compared to the control antibody group (Figure 4, "Cont. IgG-MMAE"), the polyclonal SYT2 antibody group (Figure 4, "α-SYT2 IgG-MMAE") significantly reduced axonal mass, with the relative axonal mass reaching approximately 87.15%. The SYT2 antibody group also significantly reduced axonal mass compared to the MMAE-only group (Figure 4, "MMAE"), with the axonal mass reaching approximately 82.7% of that of the MMAE-only group. No significant differences were observed between the control antibody group and the MMAE-only group.

[0239] These results suggest that by using antibodies capable of binding to the intravesicular domain of vesicular membrane proteins, molecules such as physiologically active substances bound to the antibodies can be targeted to motor neurons, and in the case of physiologically active substances, their pharmacological effects can be obtained in motor neurons.

[0240] Example 4: Verification of pharmacological effects of drug delivery using SYNGR3 antibody When an antibody capable of binding to the intravesicular domain of SYNGR3 was used, the drug-based pharmacological effects were investigated when the drug was delivered into motor neurons in the same manner as for SYT2.

[0241] The experiment was performed in the same manner as in Example 3, except that a polyclonal goat anti-SYNGR3 intravesicular domain antibody (Synaptogyrin 3 Rabbit anti-Human Polyclonal Antibody; catalog number LS-C55548-100; LS Bio) was used as the anti-SYNGR3 intravesicular domain antibody. The experiment was performed with 10 samples per group.

[0242] The results are shown in Figure 5. Figure 5 is a graph quantitatively showing the results when a polyclonal goat anti-SYNGR3 intravesicular domain antibody was used.

[0243] MMAE inhibits the polymerization of microtubules, a cytoskeleton important for axonal elongation and maintenance, so the stronger the effect of MMAE, the more axonal elongation and maintenance are inhibited, leading to a decrease in axon mass.

[0244] Although no significant difference was observed, the axonal volume tended to decrease in the polyclonal goat anti-SYNGR3 intravesicular domain antibody group ("α-SYNGR3 IgG-MMAE" in Figure 5) compared to the control antibody ("Cont. IgG-MMAE" in Figure 5).

[0245] This suggests that it is possible to deliver physiologically active substances to motor neurons by using antibodies that can bind to the intravesicular domain of SYNGR3.

[0246] Example 5: Verification of pharmacological effects of drug delivery using the monoclonal antibody of the present invention The influence of drug delivery into motor neurons using the monoclonal antibody of the present invention obtained in Example 1 on the pharmacological effects of the drug was investigated.

[0247] The experiment was performed in the same manner as in Example 4, except that the monoclonal antibody of the present invention obtained in Example 1 was used as the anti-SYNGR3 intravesicular domain antibody and a polyclonal goat anti-SYNGR3 intravesicular domain antibody was used instead of the control antibody. Six samples were used in each group.

[0248] The results are shown in Figure 6. Figure 6 is a graph quantitatively showing the results when the monoclonal antibody of the present invention was used.

[0249] Compared with the polyclonal antibody group ("Polyclonal Ab" in Figure 6), the axon amount was significantly reduced in the monoclonal SYNGR3 antibody group ("Monoclonal Ab" in Figure 6).

[0250] This suggests that when the monoclonal antibody of the present invention is used, it is possible to deliver physiologically active substances and the like to motor neurons significantly and efficiently.

[0251] Example 6: Confirmation of affinity of monoclonal antibodies of the present invention by ELISA The binding affinity of the monoclonal antibodies of the present invention obtained in Example 1 to antigens was examined by ELISA using cultured cells expressing SYNGR3 on the cell membrane.

[0252] 1. Cell Preparation SYNGR3-expressing cells were prepared as follows. The outline of the preparation procedure is as follows.

[0253] The antigen expression vector used in "7-2. Screening by flow cytometry" in Example 1 was used as the SYNGR3 expression vector.

[0254] HEK293T cells (American Type Culture Collection) prepared as follows were used as transfection cells.

[0255] Purchased HEK293 cells were thawed and resuspended in maintenance medium (DMEM medium (Thermo Fisher Scientific) supplemented with 1% Penicillin-Streptomycin (Thermo Fisher Scientific), 10% Fetal Bovine Serum (Cytiva), and 1% GlutaMAX™ I (Thermo Fisher Scientific)). The cell suspension was centrifuged at 130 × g for 5 minutes at room temperature, the supernatant was removed, and 10 mL to 20 mL of maintenance medium was added to suspend the cells. The number of cells was counted, and 2.0 × 10 cells were plated in a 10 cm dish (Greiner Bio-One). 6 The cells were seeded at a cell density of 100 cells / dish.

[0256] After culturing the cells for 3 days, the medium was removed from the 10 cm dish and washed with PBS. After washing, 2 mL of 0.1% trypsin (Nacalai Tesque) was added and incubated at room temperature for 1.5 to 2 minutes to detach the cells, which were then collected in a 50 mL tube. The collected cells were dispersed, and the resulting cell suspension was centrifuged at 130 × g at room temperature for 5 minutes to remove the supernatant. 10 mL to 20 mL of maintenance medium was added to suspend the cells. The number of cells was counted, and 1.0 × 10 cells were used for transfection.3 The cells were seeded onto a 96-well plate (Azone) at a cell density of 100 cells / well and cultured in a maintenance medium until the cells became 80 to 90% confluent.

[0257] After culturing the cells for transfection, the medium was removed and replaced with 1 mL of transfection medium. After medium replacement, 100 μL of transfection solution was added and incubated at 37°C in the presence of 5% CO for 24 hours.

[0258] The transfection solution consisted of 0.5 μL of vector solution containing a plasmid vector at a concentration of 1 μg / μL and Opti-MEM. TM I Reduced Serum Medium (Thermo Fisher Scientific) 100 μL was mixed.

[0259] The transfection medium contains 1 μL of Lipofectamine. TM Opti-MEM supplemented with 2000 Transfection Reagent (Thermo Fisher Scientific) TM I Reduced Serum Medium (Thermo Fisher Scientific) was used.

[0260] 2. ELISA Test After transfection, the medium was removed from the 96-well plate, and 2% PFA solution was added. After fixing at room temperature for 15 minutes, the cell membrane was permeabilized with PBS containing 0.25% Triton X-100.

[0261] After permeabilization, the cells were washed with PBS, and blocking buffer (PBS + 2% normal goat serum + 1% BSA + 1% fetal bovine serum + 0.02% Triton X-100) was added to each well, followed by blocking at room temperature for 1 hour.

[0262] Primary antibody solutions were prepared in 1.5 mL tubes at concentrations of 1 μg / mL, 500 ng / mL, and 100 ng / mL. Measurement medium was used as the solvent, and the primary antibody was a control polyclonal goat anti-SYNGR3 intravesicular domain antibody or a monoclonal antibody of the present invention.

[0263] After blocking, the medium was removed from the 96-well plate, and 100 μL / well of a primary antibody solution was added to each well, followed by incubation at 37° C. in the presence of 5% CO 2 for 1 hour.

[0264] After the primary antibody reaction, the antibody solution was removed and washed with PBS. A secondary antibody solution was added and incubated at room temperature for 1 hour. The secondary antibody solution was prepared using blocking buffer. Donkey anti-Goat 555 (Cat. No. #A32814; Thermo Fisher Scientific) was used as the secondary antibody against the polyclonal goat anti-SYNGR3 intravesicular domain antibody, and Goat anti-Mouse 555 (Cat. No. #A32723; Thermo Fisher Scientific) was used as the secondary antibody against the monoclonal antibody of the present invention. DAPI (Cat. No. D1306; Thermo Fisher Scientific) was also added to the secondary antibody solution for each staining. After the secondary antibody reaction, the antibody solution was removed and the cells were washed with PBS containing 0.02% Triton X-100.

[0265] 3. Detection and measurement of fluorescent signals Fluorescent signals were measured using Promega GloMax (Promega Corporation) at the following excitation and detection wavelengths: DAPI: excitation wavelength 365 nm, emission wavelength 415-445 nm, detection wavelength 475 nm Alexa 555: excitation wavelength 525 nm, emission wavelength 580-640 nm, detection wavelength 520 nm

[0266] The relative fluorescence intensity of the anti-SYNGR3 antibody was calculated by dividing the detected Alexa 488 signal by the DAPI fluorescence intensity. The relative fluorescence intensity when a control antibody was used was set to 1.0, and the normalized value was used to calculate the relative antibody amount.

[0267] The results are shown in Figures 7 and 8. Figure 7 is a graph showing the results when 100 ng / mL of anti-SYNGR3 antibody was used, and Figure 8 is a graph showing the results when 500 ng / mL of anti-SYNGR3 antibody was used.

[0268] The antibodies of the present invention had significantly higher binding ability to SYNGR3 than the control antibody, even when used at a low concentration of 100 ng / mL.

[0269] These results confirmed that the antibodies of the present invention are excellent antibodies, consistent with the results of the antibody affinity evaluation performed in Example 2. All publications, patents, and patent applications cited herein are incorporated herein by reference in their entirety.

Claims

1. An antibody capable of binding to the intravesicular domain of synaptogyrin 3, comprising a heavy chain variable region comprising an HCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 3, an HCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 4, and an HCDR3 consisting of the amino acid sequence shown in SEQ ID NO: 5, and a light chain variable region comprising an LCDR1 consisting of the amino acid sequence shown in SEQ ID NO: 6, an LCDR2 consisting of the amino acid sequence shown in SEQ ID NO: 7, and an LCDR3 consisting of the amino acid sequence shown in SEQ ID NO:

8.

2. The antibody according to claim 1, selected from any of the following (a) to (c): (a) an antibody in which the heavy chain variable region consists of an amino acid sequence having 90% or more sequence identity to SEQ ID NO: 1, and the light chain variable region consists of an amino acid sequence having 90% or more sequence identity to SEQ ID NO: 2; (b) an antibody in which the heavy chain variable region consists of an amino acid sequence in SEQ ID NO: 1 in which one or more amino acids have been substituted, deleted, and / or added, and the light chain variable region consists of an amino acid sequence in SEQ ID NO: 2 in which one or more amino acids have been substituted, deleted, and / or added; or (c) an antibody in which the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:

2.

3. A conjugate of the antibody according to claim 1 with a labeling substance and / or a physiologically active substance.

4. A nucleic acid molecule having a base sequence encoding the antibody described in claim 1.

5. A cell comprising the nucleic acid molecule of claim 4.

6. A targeting agent for motor neurons comprising the antibody described in claim 1.

7. The targeting agent of claim 6, further comprising a labeling substance and / or a biologically active substance.

8. The targeting agent of claim 7, comprising a conjugate of the antibody with the labeling substance and / or the physiologically active substance.

9. The targeting agent of claim 7, wherein the labeling substance is a fluorescent molecule.

10. The targeting agent of claim 7, comprising the labeling substance, which is a motor neuron visualization agent.

11. The targeting agent of claim 7, wherein the physiologically active substance is one or more selected from the group consisting of synapse formation promoters, synapse maintenance agents, muscle-building agents, and nerve cell function modifying agents.

12. The targeting agent of claim 6, which is taken up into a cell by endocytosis.

13. A pharmaceutical composition comprising the targeting agent of claim 7.

14. A method for targeting the labeled substance and / or the physiologically active substance to a motor neuron, comprising the steps of contacting a motor neuron with the targeting agent described in claim 7 and / or the pharmaceutical composition described in claim 13, and delivering the targeting agent and / or the pharmaceutical composition to a synapse of the motor neuron.

15. A method for visualizing motor neurons, comprising the steps of contacting a motor neuron with a targeting agent, which is the motor neuron visualization agent described in claim 10, delivering the targeting agent to a synapse of the motor neuron, and detecting a signal of the labeling substance.

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