WO2025089345A1 - Anti-synaptogyrin 3 antibody - Google Patents

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

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 a motor neuron, and a pharmaceutical composition containing the same.

The nervous system is made up of the central nervous system and peripheral nervous system, and 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).

The central and peripheral nerves are each composed of nerve cells, and these cells exchange signals with each other through synapses. A synapse is a junction that includes a gap formed between the axon terminal of a nerve cell (presynapse) and the dendrite of another nerve cell or a cell of a skeletal muscle or organ (postsynapse), and 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.

In recent years, compounds and peptides have been developed that can maintain and improve neural function by promoting synapse formation. Patent Document 1 describes that a specific peptide has the effect of promoting dendritic outgrowth and synapse formation in primary cultured cortical neuron cells (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), which is generated by cleavage of amyloid precursor protein (APP) by β-secretase, promotes synapse formation, and that CTFβ is used to treat neurodegenerative diseases, etc.

Furthermore, Patent Document 3 describes a method for culturing motor neurons with presynapses using microbeads with LRRTM molecules or fusion proteins containing said molecules immobilized on their surface. In this way, with the development of a technology that allows for easy in vitro screening of the effects of drugs on motor neuron function, it is expected that in the future a large number of compounds and drug candidates that can act on motor neurons will be provided.

JP 2012-092048 A JP 2008-143867 A WO2021/006075

The present inventors have developed a method that enables the controlled delivery of drugs to target motor neurons, using antibodies capable of binding to membrane proteins present in the synaptic vesicles of motor neurons (PCT/JP2023/016125). According to this method, it has been confirmed that it is possible to obtain the pharmacological effects of a physiologically active substance linked to an antibody in a target motor neuron, using an antibody capable of binding to the intravesicular domain of several types of vesicular membrane proteins, including synaptotagmin 2. However, because such a method of use had not been anticipated until now, no excellent antibodies capable of binding to membrane proteins that would exert a good effect using this method were known.

The objective of the present invention is therefore 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 of targeting a substance to a motor neuron and a means of visualizing the targeting site using the same.

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, thus completing the present invention.

The present invention has been 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 including an HCDR1 having the amino acid sequence shown in SEQ ID NO: 3, an HCDR2 having the amino acid sequence shown in SEQ ID NO: 4, and an HCDR3 having the amino acid sequence shown in SEQ ID NO: 5, and a light chain variable region including an LCDR1 having the amino acid sequence shown in SEQ ID NO: 6, an LCDR2 having the amino acid sequence shown in SEQ ID NO: 7, and an LCDR3 having the amino acid sequence shown in SEQ ID NO: 8.
[2] The antibody according to [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 with one or more amino acid substitutions, deletions, and/or additions, and the light chain variable region consists of an amino acid sequence in 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 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 [1] or [2] with a labeling substance and/or a physiologically active substance.
[4] A nucleic acid molecule having a base 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 one of [7] to [9], which is a motor neuron visualization agent and contains the labeling substance.
[11] The targeting agent according to any one of [7] to [10], wherein the physiologically active substance is one or more selected from the group consisting of synapse formation promoters, synapse maintenance agents, muscle strengthening agents, and nerve cell function modifying agents.
[12] The targeting agent according to any one of [6] to [11], which is taken up into a cell by endocytosis.
[13] A pharmaceutical composition comprising the targeting agent according to any one of [7] to [12].
[14] A method for targeting a labeled substance and/or a physiologically active substance to a motor neuron, comprising the steps of contacting a motor neuron 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 a synapse of the motor neuron.
[15] A method for visualizing motor neurons, comprising the steps of contacting a targeting agent, which is the motor neuron visualization agent described in [10], with a motor neuron, delivering the targeting agent to a synapse of the motor neuron, and detecting a signal of the labeling substance.
[16] A method for preventing or treating a condition or disease, comprising the steps of contacting a motor neuron 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 described in [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 one 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 pharmaceutical comprising the antibody and a physiologically active substance, wherein the antibody comprises 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.
[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 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.
[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, 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.
[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, 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.
This specification includes the disclosure of Japanese Patent Application No. 2023-182983, which is the priority basis of this application.

The present invention provides a monoclonal antibody 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. When the substance is a labeling substance, motor neurons can be visualized.

Figure 1 is a schematic diagram showing the experimental procedure for induction of presynaptic sites and delivery of antibodies using LRRTM2 beads. When neurospheres are seeded on a plate, nerve axons extend from the neurospheres. Then, by seeding LRRTM2 beads on the neurospheres, presynaptic sites are induced from the extended nerve axons on the surface of the LRRTM2 beads. A control antibody or an anti-synaptic vesicle membrane protein antibody was introduced into the plate in which the presynaptic sites had been induced, and the nerve cells were activated to examine whether the anti-synaptic vesicle membrane protein antibody was targeted to the presynaptic sites and exhibited a pharmacological effect. Figure 2 shows fluorescence images of the control antibody group, which was a conjugate of normal rabbit antibody and monomethyl auristatin E (MMAE). The fluorescence signal shown in the figure is a signal based on βIII tubulin (Tuj1). In Figure A, the black dashed circle indicates the position of the neurosphere. Figures B and C show enlarged images of the area in the white frame in Figure A. In Figure A, the scale bar indicates 1 mm, and in Figures B and C, the scale bar indicates 100 μm. Figure 3 shows fluorescence images of the SYT2 antibody group in which anti-synaptotagmin 2 intravesicular domain (N-terminus) polyclonal antibody (polyclonal anti-SYT2 N-terminus antibody) was conjugated with MMAE. The fluorescent signal shown in the figure is a signal based on Tuj1. Figures B and C show enlarged images of the area enclosed in the white frame in Figure A. In Figure A, the scale bar indicates 1 mm, and in Figures B and C, the scale bar indicates 100 μm. Figure 4 shows a graph quantifying the pharmacological effect based on MMAE. In the figure, the relative axonal mass is a standardized value, with the result when a control normal rabbit antibody and MMAE conjugate was used ("Cont. IgG-MMAE" in the figure) taken as 100%. In the figure, the dashed line indicates the position where the relative axonal mass is 100%, "MMAE" shows the result for the single-infusion group in which MMAE was introduced alone, and "α-SYT2 IgG-MMAE" shows the result for the SYT2 antibody group in which a polyclonal anti-SYT2 N-terminal antibody and MMAE conjugate were introduced. In the figure, the error bars indicate the 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 axonal volume is a standardized value, with the result when a control normal rabbit antibody and MMAE conjugate was used (in the figure, "Cont. IgG-MMAE") set as 100%. In the figure, the dashed line indicates the position where the relative axonal volume is 100%, and "α-SYNGR3 IgG-MMAE" shows the result of the SYNGR3 antibody group introduced with a polyclonal anti-SYNGR3 intravesicular domain antibody and MMAE conjugate. In the figure, the error bars indicate the standard error. Figure 6 shows a graph quantifying the pharmacological effect based on MMAE. In the figure, the relative axonal mass is a standardized value, with the result when a polyclonal anti-SYNGR3 intravesicular domain antibody and MMAE conjugate was used (in the figure, "Polyclonal Ab") taken as 100%. In the figure, the dashed line indicates the position where the relative axonal mass is 100%, and "Monoclonal Ab" shows the results for the SYNGR3 antibody group to which a conjugate of the monoclonal antibody of the present invention and MMAE was introduced. In the figure, the error bars indicate the standard error, and "***" indicates p<0.001. Figure 7 is a graph showing the quantification by ELISA of the binding affinity to synaptogyrin 3 when 100 ng/mL anti-SYNGR3 antibody was used. In the figure, the relative antibody amount is a standardized value assuming the result when 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" shows the result 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. Figure 8 is a graph showing the binding affinity to synaptogyrin 3 quantified by ELISA when 500 ng/mL anti-SYNGR3 antibody was used. In the figure, the relative antibody amount is a standardized value assuming the result when 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" shows the result 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.

The present invention provides a monoclonal antibody (referred to as the "antibody of the present invention") that can bind to the intravesicular domain of synaptogyrin 3 and can be used to target a substance of interest to a motor neuron.

<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 of motor neurons and is thought to be involved in regulated exocytosis, dopamine recycling, and the like. Synaptogyrin 3 has three cytoplasmic domains and two intravesicular domains separated by four transmembrane domains.

The vesicular domain of synaptogyrin 3 is exposed to the lumen of synaptic vesicles. When synaptic vesicles fuse with the plasma membrane as the intracellular calcium ion concentration increases, the lumen of the synaptic vesicle connects with the extracellular space, and the vesicular domain of synaptogyrin 3 is temporarily exposed to the outside of the cell. The plasma membrane portion containing synaptogyrin 3 is then retrieved into the cell as a synaptic vesicle membrane by endocytosis and reused as synaptic vesicles. At this time, the vesicular domain of synaptogyrin 3 is again exposed to the lumen of the synaptic vesicle.

Specifically, an exemplary human synaptogyrin 3 is a protein consisting of 229 amino acids whose amino acid sequence is represented by 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.

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

In this specification, the term "synaptogyrin 3 vesicular domain" refers to all or part of either of the two vesicular domains of synaptogyrin 3. The entire intravesicular domain includes, for example, 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. A portion of the intravesicular domain includes, for example, a region represented by a partial sequence of any length in SEQ ID NO: 10 and/or 11. A preferred portion of the intravesicular domain includes, for example, the amino acid sequence represented by the amino acid sequence of positions 127 to 144 in SEQ ID NO: 9 (SEQ ID NO: 12).

As for the antigen peptide used to obtain the antibody, a peptide containing additional amino acids in the amino acid sequence of the intravesicular domain or a part of it can be used, so long as it is possible to obtain an antibody that can bind to the intravesicular domain. Examples of antigen peptides that can be used in this way include peptides having the amino acid sequence shown in SEQ ID NO: 13 or 14.

<Antibody of the Invention>
The antibody of the present invention is a monoclonal antibody (referred to as "the antibody of the present invention") that can specifically recognize and bind to the intravesicular domain of synaptogyrin 3. The antibody of the present invention binds to the intravesicular domain of synaptogyrin 3, but preferably does not bind to the intravesicular domain of other membrane proteins belonging to the synaptogyrin family (e.g., synaptogyrin 1 and synaptogyrin 4). In addition, the antibody of the present invention binds 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).

In this specification, "binding" and "specific binding" are not particularly limited, but may mean that the binding between the antigen and the antibody has a binding affinity with a KD value of 10 ^-8 M or less. The antibody of the present invention has the above-mentioned binding affinity to the intravesicular domain of synaptogyrin 3. It is preferable that the antibody of the present invention does not have the above-mentioned binding affinity to other antigens including other proteins (for example, intravesicular domains of other membrane proteins belonging to the synaptotagmin family such as synaptotagmin 1, etc.). 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 × 10 ⁵ /Ms or more, 15 × 10 ⁵ /Ms or more, 16 × 10 ⁵ /Ms or more, etc.). Methods for measuring the binding affinity and binding rate constant include, for example, surface plasmon resonance and biolayer interference, and for example, biolayer interference can be preferably used.

Alternatively, in this specification, "specifically binds to a target substance" can mean that the binding to a target substance, for example, the intravesicular domain of synaptogyrin 3, is at least two-fold, three-fold, or four-fold stronger than the binding to a substance other than the target substance, for example, when detected by a general binding assay using an excess amount of the target substance. It can also mean that, when binding is detected by fluorescent labeling as described above, the S/N ratio is 2 or more, 3 or more, or 4 or more.

The antibody of the present invention may be any of non-human antibodies of mouse, rabbit, goat, etc., chimeric antibodies, humanized antibodies, and human antibodies, but when used to treat conditions and diseases in humans, it is preferable, although not limited thereto, that it be a humanized antibody or a human antibody. Therefore, an antibody that can be suitably used in the present invention may be an antibody that contains a framework region (FR) of a human antibody and a complementarity determining region (CDR) that can provide high binding affinity to the intravesicular domain of synaptogyrin 3.

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, a 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 a diabody.

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.

The antibodies of the present invention further 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 to increase stability, and conjugates bound to drugs such as anticancer drugs. In this specification, 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 the sake of convenience, unless the context is inconsistent.

The antibody of the present invention can also be synthesized as a multimer, such as a dimer, trimer, or tetramer. Furthermore, the antibody of the present invention can be a bispecific antibody 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 can be, for example, for another antigen that can be expressed by the target cell, or for a physiologically active substance and/or a labeling substance to be delivered, and can be appropriately selected depending on the purpose. When the second specificity is for another antigen that can be expressed by the target cell, i.e., the motor neuron, the use of the bispecific antibody provides more specific delivery to the target cell. Furthermore, when the second specificity is for a physiologically active substance and/or a labeling substance, the substance can be delivered to the target site as a complex by administering it together with the bispecific antibody.

Those skilled in the art can obtain the antibodies of the present invention in an appropriate form according to the intended use based on the description in this specification and common technical knowledge in the field.

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

An antibody 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.

In this case, the amino acid sequences of other regions of the present invention (e.g., amino acid sequences of FR, amino acid sequences of constant regions) 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 it is preferable that the modification is within the framework region or constant region from the viewpoint of having little effect on the binding characteristics. In addition, it is preferable that the modification does not significantly change the three-dimensional structure of the antibody.

Furthermore, examples of the antibodies of the present invention that bind to the intravesicular domain of synaptogyrin 3 include the following antibodies:
(a) An antibody having the above-mentioned HCDR1 to 3 and LCDR1 to 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.

As used herein, "sequence identity" (of amino acid sequences) refers to the percentage of matching amino acid residues out of the total number of amino acid residues in the amino acid sequences of two polypeptides being compared, when the sequences are aligned by inserting appropriate gaps into one or both sequences as necessary to maximize the number of matching amino acid residues. Sequence identity can be calculated using methods and software well known in the art.

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 consists of an amino acid sequence that has a sequence identity of 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 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 a sequence identity of 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 to SEQ ID NO: 2.

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

Here, "several" can be 5 or less, for example 2 or more, 3 or more, 4 or more, or 5. Thus, the antibody of the present invention can have 5 or less (e.g., 2 to 3) amino acid modifications in the heavy chain variable region and/or the light chain variable region of the antibody (b) above. Antibodies that are functionally equivalent to a certain antibody can be prepared by techniques well known in the art, such as site-directed mutagenesis.

As used herein, "amino acid substitution" refers to substitutions between the 20 types of amino acids that make up natural proteins. The amino acid substitutions can be, but are not limited to, conservative substitutions. Conservative substitutions refer to substitutions within a conservative amino acid group that has similar properties such as charge, side chain, polarity, and aromaticity. Examples of such substitutions include substitutions within the uncharged polar amino acid group 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).

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

It is known that after an antibody against a certain antigen is produced in the body, a process called "affinity maturation" produces 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 activity equivalent to or improved over the disclosed antibodies, specifically antibodies with improved binding affinity to the intravesicular domain of synaptogyrin 3 and functionality as a targeting agent for substance delivery. These modifications can occur within the CDR regions, in addition to within the framework and constant regions.

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 identified as an antigen, and a monoclonal antibody can be obtained by a known method. The antibody of the present invention can also be obtained synthetically 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 base sequence information of the polynucleotide encoding the antibody.

When producing an antibody by genetic engineering techniques, polynucleotides encoding the heavy and light chains can be introduced into an appropriate host cell and expressed to obtain the antibody as a recombinant protein. In this case, the polynucleotide may be either DNA or RNA, and any method used in the art can be used as appropriate for introducing the polynucleotide into the host cell. As a vector for introducing the polynucleotide into the host cell, a virus vector, a plasmid vector, a phage vector, etc. can be used as appropriate. As the host cell, for example, bacteria such as E. coli, yeast, insect cells, animal cells, etc. can be used. Here, the polynucleotides encoding the heavy and light chains can be introduced into separate vectors, or can be linked to the same vector and introduced.

For example, the antibody of the present invention can exhibit excellent physiological activity in motor neurons in the form of a conjugate with a physiologically active substance. The improvement of physiological activity based on the use of the antibody of the present invention can be confirmed, for example, by using as an indicator the results obtained by administering monomethyl auristatin E (MMAE) and the conjugate of the present invention to motor neurons in which axonal elongation and presynaptic formation have been induced by co-culture with microbeads having LRRTM molecules immobilized on the surface in advance. In this case, the targeting effect of the antibody of the present invention can be confirmed by comparing the amount of axons when a conjugate of MMAE with a polyclonal goat anti-SYNGR3 intravesicular domain antibody (Synaptogyrin 3 Rabbit anti-Human Polyclonal Antibody; catalog number LS-C55548-100; antibody of LS Bio, etc.) is used with the amount of axons when a conjugate of the antibody of the present invention is used. When the antibody of the present invention is used, for example, the amount of axons is reduced compared to when a polyclonal antibody is used. For example, a statistically significant reduction would be sufficient; specifically, the average axon volume could be 95% or less, or 90% or less, of the amount observed when a polyclonal antibody was used.

<Nucleic acid molecule>
The present invention also provides a nucleic acid molecule having a base sequence encoding the antibody of the present invention. The nucleic acid molecule of the present invention may be a nucleic acid molecule encoding any one or more of the antibodies of the present invention. The base sequence of such a nucleic acid molecule is not particularly limited. Examples include a codon-optimized base sequence and a base sequence with an initiation codon (ATG) added to the 5'-end. The nucleic acid molecule 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.

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 a cell.

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

In this specification, "expressible state" refers to the placement of a gene to be expressed downstream of a promoter under the control of the promoter.

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 capable of replicating between mammalian cells and bacteria such as E. coli.

As the viral vector, for example, a retroviral vector (including oncoretroviral vectors, lentiviral vectors, and pseudotype vectors), an adenoviral vector, an adeno-associated virus (AAV) vector, a simian virus vector, a vaccinia virus vector, a Sendai virus vector, an Epstein-Barr virus (EBV) vector, and an HSV vector can be used. A viral vector lacking replication ability so as not to self-replicate within an infected cell may also be used.

In this specification, a "promoter" refers to a gene expression regulatory region that can control the expression of a gene, etc., 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 where the gene, etc. under their expression control is expressed. A ubiquitous promoter is a promoter that controls the expression of a target gene, etc. (target gene, etc.) in all cells, i.e., the entire host individual. A site-specific promoter is a promoter that controls the expression of a target gene, etc. only in a specific cell or tissue. 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 a host cell.

Promoters are also classified into constitutively active promoters, expression-inducible promoters, and time-specifically active promoters based on the time of expression. Constitutively active promoters can constitutively express a target gene, etc. in a cell. Expression-inducible promoters can induce the expression of a target gene, etc. in a cell at any time. Time-specifically active promoters can induce the expression of a target gene, etc. in a cell only at a specific time during the developmental stage. Any of these promoters can be considered to be overexpression promoters, since they can cause excessive expression of a target gene in a host cell. The promoter contained in the gene expression vector of the present invention is preferably a constitutively active promoter, which allows for long-term persistence of the therapeutic effect.

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, an Ub promoter, and a 5' LTR promoter. In the case of a retroviral vector, the nucleic acid encoding an antibody of the present invention can be placed downstream of the 5' LTR promoter to induce its gene expression.

<Cells>
Furthermore, the present invention provides a cell capable of expressing the antibody of the present invention.
The type of the host cell of the present invention is not particularly limited. For example, the organism from which the cell originates is not particularly limited, but for example, cells derived from vertebrates described later in the targeting agent can be used as the cell of the present invention. In addition, the type of the host cell is not particularly limited. For example, it may be an ectodermal cell, a mesodermal cell, an endodermal cell, or a combination thereof. For example, it may be an epithelial tissue, a connective tissue, a cartilage tissue, a bone tissue, a blood tissue (including lymphatic tissue), a muscle tissue, a nerve tissue, or a combination thereof. In addition, the cell of the present invention may be a cell that does not have differentiation potential or a cell that has differentiation potential (for example, a multipotent cell, a pluripotent cell, etc.). Specific examples of cells that have differentiation potential include mesenchymal stem cells, hematopoietic stem cells, various cancer cell lines, neural stem cells, iPS cells, and ES cells.

The term "pluripotent stem cells" used in the present invention refers to cells that have the ability to self-replicate, can be cultured in vitro, and have the multipotency to differentiate into cells that make up an individual. Specific examples include embryonic stem cells (ES cells), pluripotent stem cells (GS cells) derived from fetal primordial germ cells, and induced pluripotent stem cells (iPS cells) derived from somatic cells, but the cells preferably used in this method are human-derived iPS cells or ES cells.

ES cells are often obtained from fertilized eggs, but they can also be obtained from sources other than fertilized eggs, such as adipose tissue, placenta, and testicular cells, and 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., WO2003/046141), and these reports can be used with appropriate reference.

iPS cells are artificial stem cells derived from somatic cells, and 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 (e.g., pluripotency and proliferation ability based on 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. There have been many reports on methods for producing iPS cells, and these reports can be referred to and modified as appropriate for use. The iPS cells that can be used in this method are preferably human-derived iPS cells, for example, human fibroblast-derived iPS cells.

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 reports can be referred to and modified as appropriate for use. Alternatively, various nerve cells induced to differentiate from human iPS cells are commercially available and can be purchased, for example, from FUJIFILM Cellular Dynamics, Inc. or ReproCell Co., Ltd.

The cell of the present invention is capable of expressing the antibody of the present invention. Preferably, the cell of the present invention comprises a nucleic acid molecule of the present invention. The cell of the present invention can comprise multiple copies of the nucleic acid molecule of the present invention.

Targeting Agents
The present invention also relates to a targeting agent for motor neurons (referred to as the "targeting agent of the present invention"), which comprises an antibody of the present invention capable of binding to the intravesicular domain of synaptogyrin 3.

As used herein, the term "targeting agent" refers to an agent for delivering a specific substance to a target. As used herein, the 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. In addition, in targeting to synapses and synaptic vesicles, the substance is taken up into the cell, particularly into synaptic vesicles, following the retrieval of synaptic vesicles by endocytosis, and is 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.

For example, the targeting agent of the present invention may be taken up into synaptic vesicles, and the transported desired substance may 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 may 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 is a substance that can be present on a cell membrane or inside a cell, and may be, for example, any of polymeric compounds such as proteins and nucleic acids, low molecular compounds such as lipids, sugars, amino acids, and nucleotides, ions such as metal ions, or atoms, etc.

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

In this specification, "motor neurons" refers to a group of nerve cells 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 nerve cells, and secondary motor neurons, which are peripheral nerve cells, but the motor neurons in this specification are secondary motor neurons.

In this specification, the term "secondary motor neuron" refers to a motor neuron that has its cell body in the anterior horn of the spinal cord or the brain stem and extends its axon to the junction with skeletal muscle. In this specification, the motor neuron includes, for example, alpha motor neuron, beta motor neuron, and gamma motor neuron. In addition to spinal nerve cells that have their cell body in the anterior nucleus of the spinal cord, some cranial nerves such as the oculomotor nerve, trochlear nerve, abducens nerve, facial nerve, and hypoglossal nerve are also included. In this specification, the motor neuron usually secretes acetylcholine as a neurotransmitter and is classified as a cholinergic neuron. However, it may be a motor neuron that secretes a neurotransmitter other than acetylcholine. In this specification, the muscle cell to which the motor neuron projects is a skeletal muscle cell.

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 in this specification broadly include muscle cells attached to bones and other muscle cells contained in skeletal muscles, such as muscle spindles. There is no particular limitation on the type of skeletal muscle, 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, eye muscles, facial muscles, tongue muscles, and stapedius muscles. Skeletal muscle cells in this specification also include cultured skeletal muscle cells, such as cells differentiated in vitro from artificial stem cells (such as iPS cells and ES cells) and/or natural stem cells (such as mesenchymal stem cells and skeletal muscle stem cells).

In this specification, the cells may be cells derived from vertebrates. Vertebrates include fish, reptiles, amphibians, birds, and mammals. Specific examples of mammals include primates (e.g., humans). The cells may also be cells derived from livestock (chickens, horses, cows, sheep, goats, pigs, etc.), pets (tropical fish, lizards, dogs, cats, rabbits, etc.), and laboratory animals (frogs, mice, rats, monkeys, etc.). The cells do not have to be derived from one type of tissue, individual, or animal species, but may be a mixture of multiple types of cells. Furthermore, there are no particular limitations on the health of the tissue and individual from which the cells are derived.

The targeting agent of the present invention allows a desired substance to be targeted to a motor neuron (e.g., the axon terminal, axon, axon hillock, cell body, dendrites, etc. of a motor neuron) via the motor neuron synapse.

In this specification, "synapse" refers to a junction that includes a gap, which is formed between the axon terminal of a nerve cell and the dendrite of another nerve cell (in the case of the central nervous system) or the cells of skeletal muscles, organs, etc. (in the case of the peripheral nervous system).

In this specification, a synapse may be a chemical synapse, such as an excitatory synapse, an inhibitory synapse, etc. In this specification, a synapse may be a synapse formed between a nerve cell and another nerve cell (e.g., a synapse formed between an axon of a nerve cell and a dendrite of another nerve cell), or a synapse formed between a nerve cell and a cell of another type (such as a muscle cell), but is preferably a synapse formed by a presynapse on a nerve cell and a postsynapse on a skeletal muscle cell (also called a "neuromuscular junction").

In this specification, the term "presynapse" refers to the enlarged portion formed at the axon terminal of a nerve cell in a synapse, and the term "postsynapse" refers to the portion facing the presynapse in a dendrite of another nerve cell or in another cell such as a skeletal muscle or organ. Additionally, the term "synaptic cleft" refers to the space between the presynapse and the postsynapse. In 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.

In this specification, "synaptic vesicles" refer to secretory vesicles present in the cytoplasm of presynaptic neurons. In this specification, synaptic vesicles include not only vesicles that contain neurotransmitters and fuse with the cell membrane in response to a stimulus to release the neurotransmitter into the synaptic cleft, but also vesicles that are retrieved into the neuron by endocytosis (including bulk endocytosis) after the release of the neurotransmitter.

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

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

In this specification, the term "labeling substance" refers to a substance that emits a signal that can detect its presence. 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 effect 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 reagents containing ¹⁴ C, ³ H, ¹²⁵ I, and the like. The photoacoustic effect refers to a phenomenon in which adiabatic expansion accompanying light absorption generates a thermoelastic wave, and this thermoelastic wave can be detected as an acoustic wave. Examples of photoacoustic effect probes include indocyanine green or its derivatives, curcumin derivatives, and choline derivatives. When the absorption characteristics of the antibody, labeling substance, and physiologically active substance used are known, it is not necessarily necessary to use a labeling substance for the photoacoustic effect. For example, a luminescent labeling substance may be detected based on the photoacoustic effect.

In this specification, the term "biologically active substance" refers to a substance that can directly or indirectly exert a physiological effect on a living organism or a cell. Examples include low molecular weight compounds that can exert a physiological effect on a target motor neuron, functional medium molecules such as peptides and aptamers, and polymeric compounds including biopolymers such as proteins such as antibodies and enzymes, and nucleic acids such as DNA and RNA. For example, drugs or prodrugs such as synapse formation promoters, synapse maintenance agents, muscle strengthening agents, or nerve cell function modifiers can be used as bioactive substances.

"Physiological effect" refers to an effect that brings about quantitative and/or qualitative changes in biological molecules such as proteins, DNA, and RNA. As a result of a physiological effect, for example, the functions and properties of living organisms, organs, tissues, cells, etc. may change. For example, effects such as promotion or inhibition of synapse formation, improvement or prevention of decline in neuronal function, or improvement or prevention of hyperactivity of nerve cells may be obtained.

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 not covalently linked (e.g., noncovalently linked) or are covalently linked. When the antibody of the present invention and the desired substance are covalently linked, 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").

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 is a substance in which the antibody of the present invention is linked to a desired substance (a labeling substance and/or a biologically active substance).

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, so long as the antibody of the present invention and the desired substance can reach the vicinity of the motor neuron in a linked state.

In this specification, the term "synapse formation promoter" refers to a drug that has the effect of promoting the formation of the presynapse and/or postsynapse. Promotion of synapse formation includes, for example, enhancing the strength of synapse connections, for example, (i) increasing the surface area and/or volume of the presynapse and/or postsynapse, (ii) increasing and/or qualitatively changing the amount, density, accumulation rate, accumulation frequency, etc. of a protein specifically expressed in the presynapse (e.g., synapsin 1 or synapsin 2, etc.), and (iii) increasing and/or qualitatively changing the amount, density, accumulation rate, accumulation frequency, etc. of a protein specifically expressed in the postsynapse (e.g., LRRTM family proteins).

In this specification, the term "synapse maintenance agent" refers to a drug that has the effect of suppressing or assisting in the degeneration of the presynapse and/or postsynapse. Synapse maintenance includes, for example, suppressing or assisting in the weakening of synaptic junction strength, for example, (i) suppressing or assisting in the reduction of the surface area and/or volume of the presynapse and/or postsynapse, (ii) suppressing or assisting in the reduction and/or qualitative change of the amount, density, accumulation rate, accumulation frequency, etc. of a protein specifically expressed in the presynapse (e.g., synapsin 1 or synapsin 2, etc.), and (iii) suppressing or assisting in the reduction and/or qualitative change of the amount, density, accumulation rate, accumulation frequency, etc. of a protein specifically expressed in the postsynapse (e.g., LRRTM family proteins).

In this specification, the term "muscle-enhancing agent" refers to a drug that has the effect of enhancing muscle or inhibiting weakening, or the effect of promoting such enhancement. The type of muscle enhancement is not particularly limited as long as the 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 constitutes muscle, such as muscle bundles, muscle fibers, myofibrils, sarcomeres, muscle cells, and/or an effect of causing a change in the expression level of a specific protein in the cells that constitute 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.

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 JP 2022-053535 (e.g., thiamine and its derivatives), and compounds disclosed in JP 2023-028848 (e.g., atropine, busulfan, chromocarb, procainamide, udenafil, propyphenazone, and their derivatives).

In this specification, the term "neuron function modifying agent" refers to an agent that has the effect of changing or promoting the function exerted by a neuron. The modification of a neuron's function is not particularly limited as long as it changes the degree and/or nature 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 the extension, retraction, and branching of neurites, and the formation and retraction of synapses).

Specific examples of 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 monomethylauristatin E (MMAE), monomethylauristatin F, and auristatin PE).

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 the formation, maintenance, degradation, branching, running, and localization of the cytoskeleton. The cytoskeleton modifying agent used herein also includes agents that modify the formation, etc. of the cytoskeleton by acting on molecules other than the cytoskeleton. The cytoskeleton includes microtubules, intermediate filaments, and actin filaments. For example, an agent that inhibits the formation and maintenance of the cytoskeleton, specifically, for example, a microtubule polymerization inhibitor, etc., can be used as a cytoskeleton modifying agent.

These drugs only need to have an effect on nerve cells, and do not need to have an effect specific to motor neurons.

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

When the targeting agent of the present invention does not include a conjugate, the desired substance preferably has a portion capable of binding to the antibody of the present invention. Specifically, for example, a desired substance that is covalently bound to another antibody capable of binding to the antibody of the present invention can be used. 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 "<Antibody of the present invention>" section, except that the antigen is the antibody of the present invention and may be a polyclonal antibody.

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. Therefore, the moiety capable of binding to the antibody of the present invention and the desired substance may be linked by a non-covalent bond, may be directly linked by a covalent bond, or may be indirectly linked via a linker or the like.

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 the antibody of the present invention.

The site at which the desired substance binds to the antibody of the present invention is not particularly limited, so 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.

The targeting agent of the present invention may contain multiple desired substances, so long as the functions of each substance are not impaired. In this case, for example, the targeting agent may contain multiple substances of the same substance, or may contain one or multiple substances that are different from each other.

Linkers that are suitable for use in the present invention may be any linkers that are suitable for use in the field. In this case, the structure and chain length of the linker may be appropriately selected within a range that does not impair the function of the resulting conjugate. The linker may be configured, for example, so that it can be cleaved after transport to the synapse. The linker may also be configured, for example, so that it cannot be cleaved after transport to the synapse.

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, may be preferably used. In addition, a cleavable linker, such as an acid-labile linker, a photolabile linker, a peptidase-sensitive linker, a dimethyl linker, or a disulfide-containing linker, may also be used.

The antibody of the present invention contained in the targeting agent of the present invention binds to the intravesicular domain of synaptogyrin 3 that is temporarily exposed on the cell surface due to fusion of synaptic vesicles with the cell membrane, and can be delivered into the cell together with synaptogyrin 3 along with endocytosis of synaptic vesicles. Therefore, it is preferable that the targeting agent or conjugate is designed so that the desired substance is delivered to the synaptic vesicles of the target synapse. The characteristics of compounds that can be delivered to synaptic vesicles are well known in the art. Since the diameter of endosomes in bulk endocytosis is 90 nm to 160 nm, the average (or median) particle size of the targeting agent or conjugate 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. In addition, since the diameter of synaptic vesicles is 40 nm to 60 nm, the average particle diameter (or median) 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, when the targeting agent does not contain the desired substance, the overall particle diameter when the desired substance is bound to the targeting agent can be in the above-mentioned range. However, for example, the particle diameter may be designed to be large for the purpose of inhibiting endocytosis of synaptic vesicles or for the purpose of delivering a substance to the synapse surface or synaptic cleft. In addition, for example, when designed so that the desired substance is already separated at the time of delivery to the synaptic vesicles, the particle diameter before separation may be designed to be large.

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

The targeting effect on motor neurons via synapses 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.

The inventors have previously found that presynaptic formation can be induced by co-culturing nerve cells with microbeads having LRRTM molecules (such as the extracellular domain of LRRTM2) immobilized on their surfaces (WO2021/006075).

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 presynapse induced by co-culturing neurons with microbeads.

"LRRTM (leucine-rich repeat transmembrane neuronal protein) family protein" 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 the formation of presynapses. In mammals, including humans, four types of LRRTM family proteins have been reported: LRRTM1, LRRTM2, LRRTM3, and LRRTM4. The LRRTM family protein used in the microbeads may be any of these.

<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.

<Visualization agent for motor neurons or their synapses>
The present invention provides a targeting agent (hereinafter referred to as the "visualizing agent of the present invention") that is an agent for visualizing motor neurons or their synapses.

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

"Visualizing a motor neuron" refers to making the entire or a part of a motor neuron detectable. When visualizing a part of a motor neuron, the part that is visualized may be random or a predetermined part. For example, a synapse can be visualized as the predetermined part. In that case, the visualization agent of the present invention can be used as a synapse visualization agent. "Visualizing a synapse" refers to making a presynapse and/or a postsynapse detectable. Therefore, the visualization agent of the present invention can use any detectable labeling substance in addition to a labeling substance that can be detected directly by visual inspection. Similarly, the visualization agent of the present invention can visualize the axon terminal, axon, axon hillock, cell body, dendrites, etc. of a motor neuron.

The visualization agent of the present invention may be used in vivo or in vitro. Detection of the signal of the labeling substance may 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 known in the field of in vivo imaging, luminescent substances such as chemo- or bioluminescent substances, sound-emitting substances such as photoacoustic probes, radioactive substances such as radioisotopes, or contrast agents.

The visualization agent 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.

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

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

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

The composition or pharmaceutical composition of the present invention includes the targeting agent of the present invention, which contains a physiologically active substance. In addition to the targeting agent, the composition or pharmaceutical composition of the present invention may also include additives (e.g., carriers (solid or liquid carriers, etc.), excipients, surfactants, binders, disintegrants, lubricants, solubilizing agents, suspending agents, coating agents, colorants, preservatives, buffers, pH adjusters), etc., as necessary. In this case, the additives can be appropriately selected depending on the dosage form of the composition or pharmaceutical composition.

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.

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

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

In this specification, "nerve damage" refers to damage at any point on a nerve, and includes damage physically inflicted from outside the body, as well as damage caused by internal factors such as cancer or tumors.

In this specification, "aging" refers to various functional declines, morphological changes, changes in appearance, etc. that occur in individual organisms over time, and the processes involved.

Frailty and sarcopenia are known as conditions caused by aging. Frailty refers to a state in which physical and mental vitality (motor function, cognitive function, etc.) declines with age, and daily life functions are impaired and the body and mind become fragile, influenced by 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. On the other hand, sarcopenia refers to a state in which skeletal muscle mass and muscle strength decrease due to aging or disease. Morphological changes such as synaptic detachment and partial or complete axonal detachment from the postsynapse have been observed in the neuromuscular junction of old mice, and it is believed that morphological changes in the neuromuscular junction associated with aging are involved in the decrease in skeletal muscle mass in sarcopenia.

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 who have or are at high risk of having frailty or sarcopenia.

In the present invention, diseases include, for example, neurological diseases and neuromuscular diseases.
As used herein, the term "neurological disease" refers to a disease caused by a disorder of nerves such as the central nervous system or peripheral nerves, and refers to, 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.

In this specification, the term "neuromuscular disease" refers to a disease caused by a disorder of either the motor nerves, the neuromuscular junction, 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.

Amyotrophic lateral sclerosis (ALS) is a disease in which primary and secondary motor nerves are selectively and progressively degenerated and lost, and it is known that the initial pathology is detachment of motor nerves 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 the formation of synapses between skeletal muscles and motor nerves or inhibit degeneration, can be used particularly to improve or prevent the decline in neurological function caused by amyotrophic lateral sclerosis.

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 contains the conjugate or targeting agent of the present invention.

The composition or pharmaceutical composition of the present invention containing a muscle-enhancing agent can improve or prevent muscle weakness by strengthening muscles. Therefore, the composition or pharmaceutical composition of the present invention containing a muscle-enhancing agent can be used to improve or prevent muscle weakness, such as weakness after trauma or surgery, weakness due to aging, or weakness due to disease, or to improve muscle function.

In this specification, "trauma" refers to damage to tissues or organs caused by external factors, and includes, for example, wounds, fractures, sprains, ruptured internal organs, burns, frostbite, etc.

The composition or pharmaceutical composition of the present invention containing a function-modifying agent can improve or prevent the decline or enhancement of nerve cells by modifying the function of motor nerve cells. Therefore, the composition or pharmaceutical composition of the present invention containing a function-modifying agent can also be used to improve or prevent nerve cell hyperactivity, for example, nerve cell 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 trauma or disease.

Specifically, in addition to the various diseases and conditions mentioned above, it 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, posture abnormalities, spasticity, hypertonia, myotonia, hyperactive deep tendon reflexes, dysphagia, etc.).

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

The composition or pharmaceutical composition of the present invention may be configured to exert a pharmacological effect in any cell other than motor neurons in which synaptogyrin 3 can be expressed. In this case, the specific target disease and condition vary depending on the target cell and are not particularly limited. For example, the target may include any of the above-mentioned diseases and conditions, 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.), circulatory diseases, blood diseases, digestive system diseases, immune system diseases (inflammatory diseases, autoimmune diseases, etc.), cancer, lifestyle-related diseases, autonomic nervous system disorders, etc.

The composition or pharmaceutical composition of the present invention may contain a plurality of targeting agents of the present invention, and may further contain other active ingredients. The other active ingredients are not particularly limited as long as they do not impair the function of the targeting agent contained in the composition or pharmaceutical composition. When a plurality of targeting agents are contained, a targeting agent capable of binding to a membrane protein other than synaptogyrin 3 may be contained. Examples of such antibodies include anti-synaptotagmin 2 antibodies (anti-synaptotagmin 2 intravesicular domain (N-terminus) antibodies, etc.). In addition, for example, even if it is synaptogyrin 3, a targeting agent containing an antibody capable of binding to a different site (e.g., a different epitope or a different intravesicular domain) may be contained. The labeling substances and/or physiologically active substances contained in these targeting agents may be different or the same.

The nucleic acid molecules and/or cells of the present invention can be included in the composition or pharmaceutical composition of the present invention.

Methods for targeting motor neurons or synapses
The present invention relates to a method for targeting a motor neuron or a synapse. According to the present invention, the antibody of the present invention that binds to the intravesicular domain of synaptogyrin 3 is brought into contact with a motor neuron, thereby allowing the antibody to be incorporated into the motor neuron (particularly into the synaptic vesicles of the motor neuron) and a desired substance (a labeling substance and/or a physiologically active substance) to be targeted 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 is targeted to 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 directly or indirectly linking the antibody to the conjugate of the present invention via a linker and bringing the conjugate into contact with the motor neuron, or by separately bringing the antibody and a desired substance that can bind to the antibody into contact with the motor neuron. Thus, in accordance with the present invention there is provided a method of targeting a motor neuron or synapse, the method comprising contacting a cell, such as a motor neuron, with an antibody (optionally linked to a substance of interest).

Method for targeting labeling substances and/or biologically active substances
The present invention relates to a method for targeting a desired substance (a labeled substance and/or a biologically active substance), comprising the steps of contacting a motor neuron with a conjugate or targeting agent of the present invention and delivering the targeting agent to the motor neuron synapse.

The subject 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 be targeted at tissue that further contains skeletal muscle cells to which motor neurons project.

The motor neurons in this method may include motor neurons of a vertebrate (non-human mammals, humans, other vertebrates). The motor neurons in this method preferably include human motor neurons. Below, the present invention will be described using human motor neurons as an example, but the method is not limited to human motor neurons.

Human motor neurons can be used without any restrictions, regardless of their origin. Examples include, but are not limited to, primary 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.

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.

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 dendrite of another neuron or in cells of skeletal muscle, organs, etc. Synapse formation can also be induced by co-culturing a cell that is to form a presynapse with a cell that is to form a postsynapse. Synapse formation can also be induced by other methods, for example, by co-culturing a motor neuron with a bead coated with the extracellular domain of LRRTM2. The step of inducing synapse formation can be performed simultaneously with or before the step of contacting the motor neuron with the targeting agent of the present invention.

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 method of contact is not particularly limited as long as the motor neurons in the sample and the targeting agent can come into contact with each other. For example, the targeting agent can be applied by directly sprinkling, spraying, dripping, or applying the targeting agent to the sample, by immersing the sample in the targeting agent, or by a combination thereof. In addition, when the sample is present in another carrier (e.g., culture medium, etc.), the targeting agent may be applied by sprinkling, spraying, dripping, or applying to the carrier.

The amount to be applied is not particularly limited, and can be set appropriately taking into consideration the number of motor neurons and other conditions. For example, the concentration of IgG antibody that can be applied is 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.

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.

The administration method is not particularly limited, but examples include local administration, enteral administration, and parenteral administration, specifically administration onto the skin, inhalation administration, enema administration, eye drops, ear drops, nasal administration, intravaginal administration, administration by tube feeding, intravenous administration, intraarterial administration, intramuscular administration, intracardiac administration, subcutaneous administration, intraosseous administration, intradermal administration, subarachnoid (cavity) administration, intraperitoneal administration, intravesical administration, transdermal administration, transmucosal administration, epidural administration, intravitreal administration, etc.

The dosage is not particularly limited and can be set appropriately 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.

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 (labeling substance and/or biologically active substance) can be used. In addition, the desired substance used does not have to be of one type either. For example, multiple types of labeling substances and/or biologically active substances can be used together or separately. Specifically, for example, a labeling substance and a biologically active substance may be used in combination.

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 method and administration method, may be the same or different each time.

When the conjugate or targeting agent of the present invention is brought into contact 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 brought into contact with a motor neuron, the motor neuron can be activated or its activity can be promoted. By activating the motor neuron or promoting its activity, the efficiency of the 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 a synaptic vesicle, it is delivered directly to the cell body via the axon by retrograde transport.

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.

Methods for spontaneous activation include, for example, placing motor neurons in an environment in which they can be activated for a sufficient period of time. Environments in which motor neurons can be activated are well known in the art.

In the method for spontaneous activation, the time for activating motor neurons (e.g., contact time with the conjugate or targeting agent of the present invention) is not particularly limited, but for example, when the method is an in vitro method, it can be 1 hour or more, 3 hours or more, 6 hours or more, 12 hours or more, 18 hours or more, or 24 hours or more. Also, when the method is an in vivo method, it can be, for example, 1 hour or more, 3 hours or more, 6 hours or more, 12 hours or more, 18 hours or more, 24 hours or more, 36 hours or more, 48 hours or more, 60 hours or more, 72 hours or more, 100 hours or more, 120 hours or more, 150 hours or more, 168 hours or more, 200 hours or more, or 240 hours or more.

Methods of activating or promoting motor neuron activity through artificial stimulation include, for example, placing motor neurons in an environment in which they can be actively active for a sufficient period of time.

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

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

The duration of the stimulation is not particularly limited, and can be set appropriately taking into consideration the type and intensity of the stimulation and other conditions. 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 for 1 hour or more.

When the method is an in vivo method, the method of activating or promoting the activity of motor neurons can be performed by a method of activating a subject (for example, a method of making a subject exercise or a method of activating brain activity, etc.) or a method of promoting the activity of motor neurons with a chemical substance, etc. Compounds that promote the activity of motor neurons include compounds used in the above-mentioned chemical stimulation, and can be administered to the subject. Compounds used in the above-mentioned chemical stimulation can be administered at a pharmaceutically acceptable concentration or method. The above-mentioned compounds can be administered to the subject so that the compound stimulation does not exert biotoxicity, but if biotoxicity is manifested, it is not necessary to administer the above-mentioned compounds to the subject. When the activity of motor neurons is promoted, the same degree of effect can usually be expected in a short time compared to the method of causing spontaneous combustion.

When a targeting agent that does not contain the desired substance is used, the desired substance and the targeting agent can be contacted with the motor neuron together or separately. When the desired substance and the targeting agent are contacted separately, the timing is not particularly limited as long as the desired substance can bind to the targeting agent. For example, the desired substance can be contacted with the motor neuron before or after the targeting agent is contacted with the motor neuron.

The method of 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.

If necessary, the method may further include a step of confirming whether or not targeting has been successful. When the targeting agent used in the method includes a physiologically active substance, it can be determined that targeting has been successful when a physiological effect is observed, for example, as described above in the section on targeting agents. Furthermore, when the targeting agent used in the method includes a labeling substance, it can be determined that targeting has been successful when a signal is detected, similar to the step of detecting the signal of the labeling substance in the visualization method described below.

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

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 an effect by itself, the effect can be exerted by placing the subject in an environment where the subject is provided with sufficient nutrition for a period of time sufficient for the physiological effect to be exerted. Also, for example, if another substance is necessary for the physiologically active substance used to exert an effect, that substance can be administered in addition.

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

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, a decline in nerve function due to aging, or a decline in nerve function due to a 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 a neuromuscular disease. In one embodiment, the targeting agent comprises a conjugate of the antibody and the physiologically active substance.

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, it may be determined based on the period it takes 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.

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

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

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 human or a non-human mammal.

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

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

For example, when the labeling substance is a fluorescent molecule or a radioactive labeling substance, a signal can be generated in the target by waiting a sufficient time for the visualization agent to be delivered to the target motor neuron synapse, that is, 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 according to the time of the step of delivering the visualization agent to the motor neuron synapse.

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, as well as adding a substance such as its substrate that is used to generate a signal.
This step can be carried out simultaneously with or prior to the step of detecting a signal, which will be described later.

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

If the labeled substance is a fluorescent substance, for example, excitation light containing light of the excitation wavelength of the labeled substance can be irradiated onto the motor neuron, and the fluorescent wavelength of the labeled substance can be detected using a detector capable of detecting the fluorescent wavelength of the labeled substance. If the labeled substance is a chemiluminescent substance, for example, it can be detected using a detector capable of detecting the emission wavelength of the labeled substance. If the labeled substance is a radioactive labeled substance, it can be detected using a detector capable of detecting the radiation emitted by the labeled substance.

Detecting the signal of the labeled substance includes detecting the presence, location, or quantity of motor neurons (e.g., synapses, cell bodies, etc.) in a sample that contains motor neurons.

In addition to the step of detecting the signal of the labeling 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 labeling substance detected in the sample by comparing it with a signal in a standard sample containing the labeling substance, or with a previously prepared reference value. There are no particular limitations on the standard sample, so long as it is a biological sample that serves as a reference for determining whether or not there is a specific condition or disease. 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 individuals 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 may be a biological sample derived from another biological species, individual, tissue, or cell. In addition, there are no particular limitations on the reference value, so long as it is a value that serves as a reference for determining whether or not there is a target condition. The reference value can be set, for example, based on the intensity or number of signals generally detected in the standard sample.

In either case, the method of comparison is not particularly limited. For example, it can be done by visual inspection, by the magnitude of the numerical values, or by statistical methods.

<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 a cell of the subject, for example, a motor neuron. When the desired substance is a physiologically active substance, the physiologically active substance can be delivered to a cell such as a motor neuron. When the desired substance is a labeling substance, the method can be used to observe the delivery site of the labeling substance (for example, a motor neuron or its synapse). The present invention also provides a conjugate of the antibody and the 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 a motor neuron or its synapse, comprising the antibody of the present invention or a conjugate of the antibody and a desired substance (a labeling substance and/or a physiologically active substance).

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 for use in the method, or a composition containing the conjugate.

The present invention provides a method for delivering a physiologically active substance to a motor neuron of 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 for use in the method, or a composition containing the conjugate.

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. Also, 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 a motor neuron or its synapse.

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

The present invention also provides use of the antibody of the present invention or the conjugate of the present invention in the manufacture of a medicine comprising the antibody and a physiologically active substance.

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

Example 1: Preparation of monoclonal antibodies
The production of monoclonal antibodies and screening using the B cell cloning method were outsourced to Medical and Biological Laboratories Co., Ltd. The outline of the production and screening procedures is as follows.

1. Synthesis of Antigen Peptides The synthesis of the following peptides (containing partial sequences of the second vesicle domain) was entrusted to Eurofins Genomics K.K. as immunization antigens and screening antigens.

The immunization antigen was bound to a carrier protein to induce an immune response using Ellman's reagent. Keyhole limpet hemocyanin (KLH) was used as the carrier protein.

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

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.

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, 5% _CO2 for 1 hour.

The cells were harvested and stained with anti-B cell marker antibodies and streptavidin (which can bind to the biotin in the screening peptide).

The stained cells were analyzed using FACS Melody (BD Japan), and cells that were positive for anti-B cell markers and biotin were isolated one by one as B cells that reacted to the antigen.

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.

Furthermore, nucleic acid fragments containing the variable region sequences were amplified using PCR from the cDNA synthesized from each cell, and agarose electrophoresis was used to confirm that genes of the desired size (VH, VL) had been amplified.

5. Construction of antibody expression vector An artificial antibody gene was constructed based on the designed gene sequences of 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". The construction of this artificial antibody gene was outsourced to Integrated DNA Technologies Co., Ltd. The amplified VH was inserted into the pcDNA3.4 plasmid together with the human IgG1 constant region (hCH) (heavy chain expression vector), and similarly, VL was inserted into the pcDNA3.4 plasmid together with the human Igκ constant region (hCL) (light chain expression vector) to construct an antibody expression vector.

6. Preparation of antibody-containing supernatant Heavy chain expression vector and light chain expression vector were introduced into HEK293T cells, and the culture supernatant 3 days after introduction was collected as antibody-containing supernatant. Lipofectamine ^TM 2000 Transfection Reagent (catalog number: #11668027, Thermo Fisher Scientific) was used for vector introduction, and Opti-MEM ^TM Reduced Serum Medium (catalog number: #31985070, Thermo Fisher Scientific) was used as the introduction medium. Note that the culture supernatant of HEK293T cells that had not been transfected was collected as a negative control supernatant.

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

7-1. Screening by ELISA The screening peptide synthesized in "1. Synthesis of antigen peptide" was added to an Immobilizer Streptavidin plate (catalog number: #436020, Thermo Fisher Scientific) on which streptavidin was immobilized to prepare an antigen-binding ELISA plate. Furthermore, antibody-containing supernatant was added as a primary antibody to this antigen-binding ELISA plate and incubated. Here, as a negative control, a negative control supernatant was added instead of the antibody-containing supernatant as the primary antibody. After incubation, an anti-mouse antibody, Anti-IgG (H+L) pAb-PE (catalog number: #330, MBL), was added as a secondary antibody and incubated.

After the secondary antibody reaction, TMB-US (catalog number: #TMB-US, Moss) was added as a color-developing substrate to the plate to develop the label, and the color reaction was stopped using H ₂ PO ₄ (catalog number: 167-02166, Fujifilm Wako Pure Chemical Industries, Ltd.).

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

Cells were collected three days after gene transfer as screening cells and subjected to flow cytometry. CytoFLEX (Beckman Coulter) was used for flow cytometry.

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 at a 200-fold dilution as the secondary antibody. As a 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.

8. Determination of antibody gene sequence One clone of antibody that showed a reaction in both ELISA and flow cytometry screening was obtained as the monoclonal antibody of the present invention, and the heavy chain expression vector and light chain expression vector used for expressing the antibody were obtained as a set of antibody expression vectors of the present invention. In order 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. The sequence determination was entrusted to Azenta Co., Ltd. and was performed by the Sanger sequencing method.

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

9. Identification of the sequences of the complementarity determining regions (CDRs) 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 the identified CDRs are shown in Table 3.

10. Preparation of purified antibody solution A set of the antibody expression vectors of the present invention was transfected into Expi CHO ^TM cells, and the culture supernatant was collected.

Antibodies were isolated from the culture supernatant using Pierce ^TM Disposable Columns, 10 mL (catalog number: #29924; Thermo Scientific). Elution with 0.1 M citrate buffer (pH = 4.0) was repeated until the antibody concentration was 0.1 mg/mL or less, and fractions with antibody concentrations of 0.1 mg/mL or more were isolated as antibody solutions. The antibody solution was dialyzed against 1x Dulbecco's phosphate buffered saline (catalog number: D1408, Sigma Aldrich) and filtered through a filter with a pore size of 0.22 μm to prepare purified antibody solutions.

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

The binding characteristics of the antibody were measured using sc-hSYNGR3 as an antigen using the Octet RED96e System (SARTORIUS). The following conditions were used for the measurement:
Biosensor: Streptavidin biosensor (catalog number: #18-5019; SARTORIUS);
Antibody concentration: 10nM/5nM/2.5nM/1.25nM/0.63nM/0.31nM/0.17nM/0nM;
Buffer: PBS containing 0.02% Tween-20 and 0.01% BSA;
Pre-immobilization equilibration: Incubate with buffer only for 60 seconds;
Immobilization: Incubate with a 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;
Both were incubated at 30° C. with an agitation speed of 1000 rpm.

　A polyclonal goat anti-SYNGR3 intravesicular domain antibody was used as a control. The antigen was used at a concentration of 0.02 μg/mL.

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.

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

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.

Example 3: Verification of pharmacological effects of drug delivery using intravesicular domain antibody
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 an antibody that binds to its intravesicular domain (N-terminus).

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 seeded in a 96-well plate (V-bottom) at a density of 2 × ¹⁰⁴ cells/well and cultured in motor neuron maintenance medium (iXCells Biotechnologies) for 1 week to produce neurospheres. The produced neurospheres were selected based on size and circularity, and those that met these criteria were used in the following experiments.

A 96-well EZVIEW® culture plate LB (AGC Technoglass Co., Ltd.) was coated with poly-D-lysine and Geltrex® Matrix (Thermo Fisher Scientific Co., Ltd.), and the selected neurospheres were seeded on the plate and cultured for 20 days. The above-mentioned motor neuron culture medium was used as the culture medium, and the medium was replaced with the same medium on the second day of culture. Thereafter, the medium was replaced three times a week with neuron medium (Neurobasal plus medium (B27 plus supplement (Thermo Fisher Scientific Co., Ltd.), 20 ng/mL BDNF, 20 ng/mL GDNF, and penicillin-streptomycin added)). All medium changes were performed at 50 μL/well.

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

Specifically, streptavidin-coated microspheres (Bangs Laboratories, Inc.; polystyrene, average diameter 9.94 μm) were washed twice with washing buffer (phosphate-buffered saline (PBS), 0.01% bovine serum albumin (BSA), 0.05% TritonX-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 washing buffer (anti-human IgG Fc antibody beads).

Then, 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 thereto, and LRRTM2-Fc was immobilized on the anti-human IgGFc antibody beads. The resulting beads were washed with washing buffer and suspended in binding buffer (LRRTM2 bead suspension).

3. Presynaptic induction (Figure 1)
After 20 days of culture, LRRTM2 beads were seeded at 0.1 μg/well on the plate and cultured at 37° C. for 48 hours to induce the formation of presynapses.

4. Preparation of conjugates The drug used was monomethyl auristatin E (MMAE: MedChemExpress), a microtubule polymerization inhibitor. Conjugates of MMAE and antibodies were prepared using the MagicLink ^TM kit (BroadPharm). The conjugates were prepared according to the manufacturer's protocol.

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).

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

As an additional control, some samples were loaded with MMAE alone rather than the conjugate (number of samples: 13). In these cases, the MMAE solution was prepared as described above, using the same concentration of MMAE instead of the conjugate.

6. Introduction of Conjugates In the plate in 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.

After the transfection, the solution was collected and washed, and the medium was replaced with neuronal medium that did not contain the conjugate or MMAE. Then, the cells were cultured for another 24 hours to allow the axons to grow.

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

9. Immunocytochemical staining Immunocytochemical staining was performed using the added antibody as the primary antibody. After permeabilization and blocking of the cell membrane of the fixed cells with a detergent, primary antibody reaction was performed using mouse anti-βIII tubulin (Tuj1) antibody (catalog number 801202; Biolegend) as an additional primary antibody. Then, secondary antibody reaction was performed using Alexa 555-labeled anti-mouse antibody (catalog number A32727; Thermo Fisher Scientific), and fluorescent images were obtained. Blocking was performed using blocking buffer (PBS + 2% normal goat serum + 1% BSA + 1% fetal bovine serum + 0.02% TritonX-100). The number of samples was as follows: MMAE alone-injected group, 13 samples; control antibody group, 17 samples; polyclonal SYT2 antibody group, 27 samples.

Fluorescence images were acquired using an inverted live cell (DMi8) microscope 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.

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

10. Analysis of axonal volume The 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 when the control normal rabbit antibody and MMAE conjugate was used was set as 100%, and the normalized value was calculated as the relative axonal volume. Brightness was acquired using LAS X software (Leica).

11. Statistical analysis Statistical analysis was performed by t-test using GraphPad Prism9 (GraphPad Software). The significance level was p<0.05.

The results are shown in Figures 2 to 4. Figures 2 and 3 are immunocytochemical staining images showing the appearance of axons in the control antibody group, which used a conjugate of a control normal rabbit antibody and MMAE (Figure 2), and the SYT2 antibody group, which used a conjugate of a polyclonal anti-SYT2 N-terminal antibody (Figure 3). Figure 4 is a graph that quantitatively shows the results.

Under all experimental conditions, axons extended from the neurospheres, and presynaptic sites were induced normally (Figs. 2A and 3A).

MMAE inhibits the polymerization of microtubules, a cytoskeleton important for the growth and maintenance of axons. Therefore, the stronger the effect of MMAE, the more axon growth and maintenance is inhibited, and the more axon mass is expected to decrease.

In the control antibody group, many thick axons and presynaptic regions observed as bulges were observed even in the distal area far from the neurosphere (Fig. 2B). In addition, in the proximal area relatively close to the neurosphere, axons were observed running in an almost parallel alignment (Fig. 2C).

On the other hand, in the SYT2 antibody group, only a few thin axons were observed in the distal area far from the neurosphere, and presynaptic formation was also poor (Figure 3B). Furthermore, in the proximal area relatively close to the neurosphere, the axonal trajectories were not as well aligned as in the control antibody group, and the axonal trajectories appeared to be disorganized (Figure 3C).

The quantitative results also supported the observations. Compared to the control antibody group ("Cont. IgG-MMAE" in Figure 4), the axonal volume was significantly reduced in the polyclonal SYT2 antibody group ("α-SYT2 IgG-MMAE" in Figure 4), with the relative axonal volume being approximately 87.15%. The results for the SYT2 antibody group were also significantly reduced compared to the single-infusion group in which MMAE was introduced alone ("MMAE" in Figure 4), with the axonal volume being approximately 82.7% of that in the single-infusion group. No significant differences were observed between the single-infusion group and the control antibody group.

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

Example 4: Verification of pharmacological effects of drug delivery using SYNGR3 antibody
In the case of using an antibody capable of binding to the intravesicular domain of SYNGR3, the pharmacological effects of drugs were examined when drugs were delivered into motor neurons in the same manner as for SYT2.

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.

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

MMAE inhibits the polymerization of microtubules, a cytoskeleton important for the growth and maintenance of axons. Therefore, the stronger the effect of MMAE, the more axon growth and maintenance is inhibited, and the more axon mass is expected to decrease.

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

This suggests that it may be possible to deliver physiologically active substances to motor neurons by using antibodies that can bind to the intravesicular domain of SYNGR3.

Example 5: Verification of pharmacological effects of drug delivery using the monoclonal antibody of the present invention
When a drug is delivered into a motor neuron using the monoclonal antibody of the present invention obtained in Example 1, the influence on the pharmacological effect of the drug was examined.

The experiment was conducted 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. The experiment was conducted with six samples per group.

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.

Compared to the polyclonal antibody group ("Polyclonal Ab" in Figure 6), the axon mass was significantly reduced in the monoclonal SYNGR3 antibody group ("Monoclonal Ab" in Figure 6).

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

Example 6: Confirmation of affinity of the monoclonal antibody of the present invention by ELISA method
The binding affinity of the monoclonal antibody of the present invention obtained in Example 1 to an antigen was examined by ELISA using cultured cells expressing SYNGR3 on the cell membrane.

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

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

HEK293T cells (American Type Culture Collection) were used for transfection as prepared as follows:

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% GlutaMAXTM I (Thermo Fisher Scientific)). The cell suspension was centrifuged at 130 × g for 5 minutes at room temperature to remove the supernatant, and 10 mL to 20 mL of maintenance medium was added to suspend the cells. The number of cells was counted and the cells were seeded at a cell density of 2.0 × ¹⁰⁶ cells/dish in a 10 cm dish (Greiner Bio-One).

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, and 10 mL to 20 mL of maintenance medium was added to suspend the cells. The number of cells was counted, and for transfection, the cells were seeded in a 96-well plate (Azone) at a cell density of 1.0 × ¹⁰³ cells/well and cultured in maintenance medium until 80 to 90% confluent.

After culturing the cells for transfection, the medium was removed and replaced with 1 mL of medium for transfection. After medium replacement, 100 μL of the transfection solution was added and incubated at 37° C. in the presence of 5% CO ₂ for 24 hours.

A transfection solution was prepared by mixing 0.5 μL of a vector solution containing a plasmid vector at a concentration of 1 μg/μL with 100 μL of Opti-MEM ^™ I Reduced Serum Medium (Thermo Fisher Scientific).

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

2. ELISA Test After transfection, the medium was removed from the 96-well plate, and 2% PFA solution was added to fix the cells at room temperature for 15 minutes. Then, the cell membrane was permeabilized with PBS containing 0.25% TritonX-100.

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

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

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.

After the primary antibody reaction, the antibody solution was removed and washed with PBS, and the secondary antibody solution was added and incubated at room temperature for 1 hour. The secondary antibody solution was prepared using blocking buffer as a solvent. Donkey anti-Goat 555 (catalog number #A32814; Thermo Fisher Scientific) was used as the secondary antibody against the polyclonal goat anti-SYNGR3 intravesicular domain antibody, and Goat anti-Mouse 555 (catalog number #A32723; Thermo Fisher Scientific) was used as the secondary antibody against the monoclonal antibody of the present invention. DAPI (catalog number 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 sections were washed with PBS containing 0.02% TritonX-100.

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 365nm, Emission wavelength 415nm-445nm, Detection wavelength 475nm
Alexa 555: Excitation wavelength 525nm, Emission wavelength 580nm-640nm, Detection wavelength 520nm

The fluorescence intensity of the detected Alexa 488 signal was divided by the fluorescence intensity of DAPI to obtain the relative fluorescence intensity of the anti-SYNGR3 antibody. Furthermore, the relative fluorescence intensity when the control antibody was used was standardized to 1.0 to obtain the relative antibody amount.

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.

The antibody 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.

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

Claims (15)

An antibody capable of binding to the intravesicular domain of synaptogyrin 3,
The antibody 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. The antibody of claim 1, which is 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 in which one or more amino acids have been substituted, deleted, and/or added, and the light chain variable region consists of the amino acid sequence of 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. A conjugate of the antibody according to claim 1 with a labeling substance and/or a biologically active substance. A nucleic acid molecule having a base sequence encoding the antibody of claim 1. A cell containing the nucleic acid molecule of claim 4. A targeting agent for motor neurons comprising the antibody of claim 1. The targeting agent according to claim 6, further comprising a labeling substance and/or a biologically active substance. The targeting agent according to claim 7, comprising a conjugate of the antibody with the labeling substance and/or the physiologically active substance. The targeting agent of claim 7, wherein the labeling substance is a fluorescent molecule. The targeting agent according to claim 7, which contains the labeling substance and is a motor neuron visualization agent. The targeting agent according to claim 7, wherein the physiologically active substance is one or more selected from the group consisting of synapse formation promoters, synapse maintenance agents, muscle strengthening agents, and nerve cell function modifiers. The targeting agent of claim 6, which is taken up into a cell by endocytosis. A pharmaceutical composition comprising the targeting agent according to claim 7. A method for targeting the labeled substance and/or the physiologically active substance to a motor neuron, comprising the steps of contacting the targeting agent of claim 7 and/or the pharmaceutical composition of claim 13 with the motor neuron, and delivering the targeting agent and/or the pharmaceutical composition to a synapse of the motor neuron. A method for visualizing motor neurons comprising the steps of contacting a targeting agent, which is the motor neuron visualization agent according to claim 10, with a motor neuron, delivering the targeting agent to a synapse of the motor neuron, and detecting a signal of the labeling substance.

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