Pharmaceutical composition for use in treatment of CADM1-related diseases and use thereof

Abstract

Provided is a pharmaceutical composition including CADM1 antibodies or antigen-binding fragments thereof. A pharmaceutical composition of the present disclosure for use in the treatment of CADM1-related diseases includes antibodies against CADM1 or antigen-binding fragments thereof.

Description

Pharmaceutical compositions for treating CADM1-related diseases and their uses

[0001] The present disclosure relates to pharmaceutical compositions and uses thereof for the treatment of CADM1-associated diseases.

[0002] Cell adhesion molecule 1 (CADM1) is expressed in various cells, including tumor cells, and is known to be involved in SARS-CoV-2 infection (Patent Document 1).

[0003] Patent No. 7270944

[0004] As a result of extensive research, the present inventors have found that anti-CADM1 antibodies can be used to treat allergic diseases such as atopic dermatitis, neurological disorders, bone diseases, type I diabetes, peripheral neuralgia, pruritus, and the like.

[0005] Therefore, a first object of the present disclosure is to provide a pharmaceutical composition comprising an anti-CADM1 antibody or an antigen-binding fragment thereof.

[0006] The present inventors also found that the use of anti-CADM1 antibodies enables transnasal intracranial delivery of drugs, delivery of drugs into bones or peripheral nerves, and delivery of drugs to mast cells or osteoblasts.

[0007] Therefore, a second object of the present disclosure is to provide a composition for delivering a drug comprising an anti-CADM1 antibody or an antigen-binding fragment thereof.

[0008] To achieve the first objective, the pharmaceutical composition of the present disclosure for use in treating a CADM1-associated disease comprises an antibody against cell adhesion molecule 1 (CADM1) (anti-CADM1 antibody) or an antigen-binding fragment thereof.

[0009] To achieve the second object, the composition of the present disclosure for use in intracranial delivery via nasal administration comprises an anti-CADM1 antibody or an antigen-binding fragment thereof and a drug.

[0010] The compositions of the present disclosure for use in intraosseous or peripheral nerve delivery comprise an anti-CADM1 antibody or antigen-binding fragment thereof and a drug.

[0011] The composition of the present disclosure for use in delivery to mast cells or osteoblasts comprises an anti-CADM1 antibody or antigen-binding fragment thereof and a drug.

[0012] The present disclosure provides a pharmaceutical composition comprising an anti-CADM1 antibody or an antigen-binding fragment thereof, and a composition for drug delivery comprising an anti-CADM1 antibody or an antigen-binding fragment thereof.

[0013] FIG. 1 is a photograph showing the results of SDS-PAGE in Example 1. FIG. 2 is a graph showing the results of ELISA in Example 1. FIG. 3 is a photograph showing the results of SDS-PAGE in Example 1. FIG. 4 is a graph showing the results of ELISA in Example 1. FIG. 5 is a graph showing the results of ELISA in Example 3. FIG. 6 is a graph showing the results of ELISA in Example 3. FIG. 7 is a schematic diagram illustrating the collected fractions in Example 4. FIG. 8 is a photograph showing the results of Western blotting in Example 4. FIG. 9 is a schematic diagram showing neuron-mast cell interaction mediated by CADM1 in Example 4. FIG. 10 is a schematic diagram showing an overall view of the preparation of an atopic dermatitis model and antibody administration in Example 4. FIG. 11 is a photograph of dermatitis in the atopic dermatitis model in Example 4. FIG. 12 is a photograph showing CADM1 expression in the nasal mucosa in Example 4. FIG. 13 is a photograph showing the results of immunostaining of the forehead of a mouse in Example 4. FIG. 14 is a microscopic image showing the nasal cavity region and the rhinencephalon region in the frozen sections of the control group and the anti-CADM1 antibody-administered group in Example 4. FIG. 15 is a photograph showing the results of Western blotting of proteins in the nasal cavity and rhinencephalon in mice to which the anti-CADM1 antibody was nasally administered in Example 4. FIG. 16 is a schematic diagram showing nose-to-brain delivery in Example 4. FIG. 17 is a photograph showing the results of TUNEL staining in Example 4. FIG. 18 is a photograph showing the results of immunostaining in Example 4. FIG. 19 is a photograph showing the results of immunostaining in Example 4. FIG. 20 is a photograph showing the results of immunostaining in Example 4. FIG. 21 is a photograph showing the results of Western blotting in Example 4. FIG. 22 is a photograph showing the results of Western blotting in Example 4. FIG. 23 is a photograph showing the results of fluorescein fluorescence observation in Example 4. Figure 24 shows a schematic diagram of an antibody-drug conjugate based on an anti-CADM1 antibody in Example 4. Figure 25 shows the results of observing the number of CADM1-expressing cells over time in Example 4.Figure 26 is a schematic diagram showing the h3E1C antibody-mouse IgG1 complex in Example 4. Figure 27 is a photograph showing the results of Western blotting in Example 4. Figure 28 is a photograph showing the results of immunostaining in Example 4. Figure 29 is a schematic diagram showing the presumed mechanism of action of the anti-CADM1 antibody in Example 4. Figure 30 is a photograph showing the results of Western blotting in Example 4. Figure 31 is a photograph showing the results of Western blotting in Example 4. Figure 32 is a photograph showing the results of Western blotting in Example 4. Figure 33 is a photograph showing the results of Western blotting in Example 4. Figure 34 is a photograph showing the results of Western blotting in Example 4. Figure 35 is a graph showing the pain-related behavior time in Example 5. Figure 36 is a graph showing the pain-related behavior time in each phase in Example 5. Figure 37 is a graph showing the pain-related behavior time in Example 5. Figure 38 is a photograph showing the administration site of the 3E1 antibody in Example 5. Figure 39 is a photograph showing a tissue section of the administration site in Example 5. Figure 40 is a photograph of a fluorescent image showing the localization of an anti-CADM1 antibody in Example 5. Figure 41 is a photograph showing a fluorescent image and a phase-contrast image of ICG in peripheral tissues in Example 5. Figure 42 is a graph and photograph showing tumor size in Example 6. Figure 43 is a graph showing the relative value of cytotoxic activity in Example 7. Figure 44 is a schematic diagram showing an overall image of the preparation of an atopic dermatitis model and antibody administration in Example 8. Figure 45 is a graph showing the relative value of dermatitis score in each group in Example 8. Figure 46 is a graph showing the relative value of scratching behavior time in each group in Example 8. Figure 47 is a graph showing the proportion of individuals who have not developed type 1 diabetes in Example 9. Figure 48 is a graph showing blood glucose levels in Example 9.

[0014] <Definition> As used herein, "CADM1" (cell adhesion molecule 1) is an intercellular adhesion molecule belonging to the immunoglobulin superfamily cell adhesion molecule group (IgCAM), and is known to transmit signals intracellularly via molecules that bind to the intracellular domain. CADM1 is also known as IgSF4A, RA175, sgIGSF, SynCAM, or Necl2. Human CADM1 is registered in UniProt, for example, under accession number Q9BY67 (see: https: / / www.uniprot.org / uniprotkb / Q9BY67 / ). Furthermore, human CADM1 has been registered as mRNA in Genbank under the accession numbers: XM_005271494.4, XM_047426695.1, XM_017017457.3, XM_047426691.1, XM_047426690.1, XM_047426693.1, XM_047426692.1, XM_047426694.1, etc. Human CADM1 has been registered as a protein or its precursor protein under the following accessions: XP_005271551.1, XP_047282651.1, XP_016872946.1, XP_047282647.1, XP_047282646.1, XP_047282649.1, and XP_047282650.1. Specific examples of human CADM1 include a protein consisting of the amino acid sequence of SEQ ID NO: 1 (Genbank ID: NP_001287974.1). Mouse CADM1 has been registered in UniProt under the accession number Q8R5M8 (see: https: / / www.uniprot.org / uniprotkb / Q9BY67 / ). Mouse CADM1 is registered as mRNA in Genbank under the accession numbers NM_001025600.1, NM_001310841.1, NM_018770.3, NM_207675.2, NM_207676.2, and the like.Furthermore, mouse CADM1 has been registered as, for example, a protein or its precursor protein under the following accessions: NP_001020771.1, NP_001297770.1, NP_061240.3, NP_997558.2, and NP_997559.1. The CADM1 may be, for example, a protein having an amino acid sequence represented by a SEQ ID NO: or an accession number, a nucleic acid encoding the protein, a functionally active mutant thereof, or a fragment thereof. A specific example of the mouse CADM1 is a protein consisting of the amino acid sequence of SEQ ID NO: 2 (Genbank ID: AB092414.1).

[0015] Human CADM1 (SEQ ID NO: 1, NP_001287974.1) MASVVLPSGSQCAAAAAAAAPPGLRLRLLLLLFSAAALIPTGDGQNLFTKDVTVIEGEVATISCQVNKSDDSVIQLLNPNRQTIYFRDFRPLKDSRFQLLNFSSSELKVSLTNVSISDEGRYFCQLYTDPPQESYTTITVLVPPRNLMIDIQKDTAVEGEEIEVNCTAMASKPATTIRWFKGNTELKGKSEVEEWSDMYTVTSQLMLKVHKEDDGVPVICQ VEHPAVTGNLQTQRYLEVQYKPQVHIQMTYPLQGLTREGDALELTCEAIGKPQPVMVTWVRVDDEMPQHAVLSGPNLFINNLNKTDNGTYRCEASNIVGKAHSDYMLYVYD TTATTEPAVHGLTQLPNSAEELDSEDLSDSRAGEEGSIRAVDHAVIGGVVAVVVFAMLCLLIILGRYFARHKGTYFTHEAKGADDAADADTAIINAEGGQNNSEEKKEYFI

[0016] Mouse CADM1 (SEQ ID NO: 2, AB092414.1) MASAVLPSGSQCAAAAAVAAAAAPPGLRLRLLLLLLSAAALIPTGDGQNLFTKDVTVIEGEVATISCQVNKSDDSVIQLLNPNRQTIYFRDFRPLKDSRFQLLNFSSSELKVSLTNVSISDEGRYFCQLYTDPPQESYTTITVLVPPRNLMIDIQKDTAVEGEEIEVNCTAMASKPATTIRWFKGNKELKGKSEVEEWSDMYTVTSQLMLKVHKEDDGVPVICQVEHPAVTGNLQTQRYLEVQYKPQVHIQMTYPLQGLTREGDAFELTCEAIGKPQPVMVTWVRVDDEMPQHAVLSGPNLFINNLNKTDNGTYRCEASNIVGKAHSDYMLYVYGT

[0017] As used herein, "anti-CADM1 antibody" refers to an antibody that binds to CADM1. The anti-CADM1 antibody is preferably an antibody that specifically binds to CADM1. The anti-CADM1 antibody can be prepared, for example, by immunizing a mammal or bird with CADM1 or a peptide thereof.

[0018] As used herein, the terms "protein," "polypeptide," "oligopeptide," and "peptide" are used interchangeably to refer to a polymer composed of unmodified amino acids (natural amino acids), modified amino acids, and / or artificial amino acids. The protein may be any length. In the protein, any amino acid in the amino acid sequence may be L- or D-type. The modified amino acid in the protein may be, for example, an amino acid that undergoes chemical modifications in vivo. Examples of such in vivo modifications include N-terminal modifications such as acetylation and myristoylation; C-terminal modifications such as amidation and glycosylphosphatidylinositol addition; and side chain modifications such as phosphorylation and glycosylation.

[0019] As used herein, "polynucleotide," "oligonucleotide," or "nucleic acid" refers to a polymer of deoxyribonucleotides (DNA), ribonucleotides (RNA), and / or modified nucleotides. The polynucleotide may be a single-stranded or double-stranded nucleic acid molecule. The polynucleotide may be composed of naturally occurring nucleotides, modified or artificial nucleotides, or both.

[0020] As used herein, the term "gene" refers to a factor that determines a genetic trait, and may refer to a "polynucleotide," an "oligonucleotide," and a "nucleic acid."

[0021] As used herein, a "corresponding" amino acid or nucleic acid refers to an amino acid or nucleotide in a given polypeptide or polynucleotide that has, or is predicted to have, the same function as a given amino acid or nucleotide or portion in the polypeptide or polynucleotide to which it is being compared.

[0022] As used herein, "identity" refers to the degree of identity when sequences to be compared (e.g., amino acid sequences or nucleotide sequences) are appropriately aligned, and refers to the percentage of exact amino acid matches between the sequences. The identity can be calculated using analysis software such as BLAST or FASTA with default parameters (the same applies hereinafter).

[0023] As used herein, "antigen" means a molecule or portion of a molecule capable of being bound by a selective binding agent, such as an antibody or an antigen binding protein, such as a T-cell receptor.

[0024] As used herein, "epitope" refers to a site bound by an antigen-binding protein such as an antibody or a T-cell receptor. The epitope can also be referred to as a region of an antigen that is bound by an antigen-binding protein that targets that antigen. The epitope can also be referred to as, for example, an antigenic determinant.

[0025] As used herein, the term "antibody" refers to a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or fragments of immunoglobulin genes, and includes a molecule or molecules capable of binding to a specific epitope on an antigen. The immunoglobulin genes include genes encoding constant regions such as κ, λ, α (including α1 and α2), γ (including γ1, γ2, γ3, and γ4), δ, ε, and μ, as well as genes encoding numerous immunoglobulin variable regions such as V, D, and J regions. The antibody comprises, for example, a heavy chain and a light chain. The light chain includes κ and λ, constituting the κ chain and the λ chain, respectively. The heavy chain includes γ, μ, α, δ, or ε, constituting the immunoglobulin classes IgG (e.g., IgG1, IgG2, IgG3, or IgG4), IgM, IgA, IgD, and IgE, respectively. The antibody may be a typical immunoglobulin (antibody) structural unit composed of a tetramer. In this case, the antibody is composed of two identical pairs of polypeptide chains, each pair consisting of one light chain (about 25 kDa) and one heavy chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100-110 or more amino acids that is primarily responsible for antigen recognition. When the antibody is derived from a chicken, the class of the antibody may be IgY.

[0026] As used herein, "heavy chain" refers to a full-length heavy chain or a fragment thereof having sufficient variable region sequence to confer binding specificity, and "light chain" refers to a full-length light chain or a fragment thereof having sufficient variable region sequence to confer binding specificity.

[0027] As used herein, the term "variable region" refers to a portion of an antibody heavy chain, typically comprising approximately 120-130 amino acids at the amino terminus of the heavy chain, or a portion of an antibody light chain, typically comprising approximately 100-110 amino acids at the amino terminus of the light chain. The variable region of an antibody typically determines the specificity of an antibody for a target antigen.

[0028] As used herein, "complementarity-determining region" (CDR) refers to a region in the variable region of an immunoglobulin (antibody) that forms an antigen-binding site. The CDR can also be referred to as, for example, a hypervariable region. The CDR is generally a region in the variable region of an antibody that is particularly highly variable in its primary structure, and is usually separated into three regions in the primary structure. In the antibody, the heavy chain variable region (HVCR) and the light chain variable region (LVCR) each contain three CDRs (heavy chain CDR1 (H1), heavy chain CDR2 (H2), and heavy chain CDR3 (H3) from the N-terminus, and light chain CDR1 (L1), light chain CDR2 (L2), and light chain CDR3 (L3) from the N-terminus). These regions are adjacent to each other in the three-dimensional structure and determine the specificity for the antigen to which they bind. Herein, the CDRs of an antibody may be determined according to the Kabat numbering system (Kabat et al., "Sequences of Proteins of Immunological Interest", 1987, US Department of Health and Human Services, NIH, USA). Alternatively, herein, the CDRs of an antibody may be determined using the definition by Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins", J. Mol. Biol., 1987;196:901-917). The variable region other than the CDRs is called the framework region (FR), which consists of FR1, FR2, FR3, and FR4 from the N-terminus, and is relatively well conserved among antibodies (Kabat et al., "Sequence of Proteins of Immunological Interest", US Dept. Health and Human Services, 1983).

[0029] As used herein, the term "antigen-binding fragment" refers to, for example, a polypeptide comprising a portion of an antibody, more specifically, a polypeptide comprising the variable region. The antigen-binding fragment can be produced, for example, by digesting the full-length immunoglobulin with various peptidases.

[0030] As used herein, the form of the "antibody" may be a full-length immunoglobulin (an antibody having an Fc region and a Fab region, a full-length antibody), or may be F(ab')2, Fab', Fab, an Fv antibody (variable fragment of antibody), a disulfide-linked Fv (dsFv), a single-chain antibody (scFv), or a polymer thereof (e.g., a diabody). The antibody may be a monoclonal antibody or a polyclonal antibody. The antibody may be, for example, a chimeric antibody, a humanized antibody, or a fully humanized antibody. The antibody may be, for example, a bispecific or oligospecific antibody.

[0031] As used herein, "F(ab') ２ The "antibody" is, for example, an antibody that contains two regions corresponding to Fab among fragments obtained by treating an antibody containing an Fab region and an Fc region with the protease pepsin. ２ can be obtained, for example, by treating an antibody against the spike protein of SARS-CoV-2 of the present disclosure (hereinafter also referred to as "S protein antibody"), which includes a Fab region and an Fc region, with the protease pepsin.

[0032] As used herein, a "Fab' antibody" refers to, for example, an F(ab') ２ The Fab' antibody is an antibody obtained by cleaving the disulfide bond in the hinge region of an antibody. ２ The antibody can be obtained by treating it with the reducing agent dithiothreitol.

[0033] As used herein, the term "Fab antibody" refers to an antibody fragment obtained by treating an antibody comprising a Fab region and an Fc region with the protease papain, in which approximately the N-terminal half of the heavy chain and the entire light chain are bound via some disulfide bonds. The Fab antibody can be obtained, for example, by treating an S protein antibody of the present disclosure comprising a Fab region and an Fc region with the protease papain.

[0034] As used herein, an "Fv antibody" is an antibody that contains an antigen-recognition site. This region comprises a dimer of one heavy-chain and one light-chain variable domain in a non-covalent association. In this configuration, the three CDRs of each variable domain can interact to form an antigen-binding site on the surface of the VH-VL dimer.

[0035] As used herein, a "disulfide-linked Fv" (dsFv) refers to an antibody in which polypeptides having cysteine ​​residues introduced into the VH and VL are linked via a disulfide bond between the cysteine ​​residues. The position at which the cysteine ​​residue is introduced can be selected based on the three-dimensional structure of the antibody, for example, by referring to the method described by Reiter et al. (Reiter et al., "Engineering interchain disulfide bonds into conserved framework regions of Fv fragments: improved biochemical characteristics of recombinant immunotoxins containing disulfide-stabilized Fv", Protein Eng. 1994 May;7(5):697-704).

[0036] As used herein, the term "scFv antibody" refers to an antibody in which VH and VL are linked via a peptide linker. The scFv antibody can be produced, for example, by obtaining cDNA encoding the VH and VL of an S protein antibody of the present disclosure, constructing a polynucleotide encoding VH-peptide linker-VL, inserting the polynucleotide into a vector, and using an expression cell.

[0037] As used herein, a "diabody" refers to an antibody having bivalent antigen-binding activity. The bivalent antigen-binding activities may be the same antigen-binding activity, or one of the two may be a different antigen-binding activity. The diabody can be produced, for example, by constructing a polynucleotide encoding an scFv so that the length of the amino acid sequence of the peptide linker is 8 residues or less, incorporating the resulting polynucleotide into a vector, and using an expression cell.

[0038] As used herein, a "monoclonal antibody" refers to an antibody in which the individual antibodies constituting a population correspond to substantially a single epitope or are substantially identical, except for antibodies with minor naturally occurring mutations. The monoclonal antibody may be prepared, for example, by the hybridoma method described in Kohler G, Milstein C., Nature. 1975 Aug. 7;256(5517):495-497 or a method similar thereto, or by phage display using a phage antibody library using a method similar to the technique described in Clackson et al., Nature. 1991 Aug. 15;352(6336):624-628, or Marks et al., J. Mol. Biol. 1991 Dec. 5;222(3):581-597.

[0039] As used herein, the term "polyclonal antibody" refers to an antibody containing a mixture of antibodies against multiple epitopes. The polyclonal antibody can be generated, for example, by administering an immunogen containing an antigen of interest to an animal or the like. The immunogen may be administered in combination with one or more immunostimulants or adjuvants. The adjuvant is used, for example, to enhance the immune response, and may include, for example, complete or incomplete Freund's adjuvant; mineral gels such as aluminum hydroxide; surfactants such as squalane, squalene, and lysolecithin; etc. Protocols for administering the immunogen are known in the art and can be carried out by any method that induces an immune response in accordance with the selected host organism.

[0040] As used herein, the term "chimeric antibody" refers to an antibody in which at least one of the heavy and light chains is composed of a variable region derived from a non-human animal immunoglobulin and a constant region derived from a human immunoglobulin, or an antibody composed of a variable region derived from a human immunoglobulin and a constant region derived from a non-human animal immunoglobulin. Chimeric antibodies can be prepared, for example, by genetic engineering. Specifically, when preparing a chicken-human chimeric antibody, the chimeric antibody can be prepared by linking a chicken leader sequence and variable region sequence to a sequence encoding a human antibody constant region (e.g., Nahoko Nishibori et al., "Expression vectors for chicken-human chimeric antibodies," Biologicals. 2004 December;32(4): pp. 213-8). Specifically, the preparation method can involve linking a chicken leader sequence and variable region sequence present in a cloned cDNA to a sequence encoding a human antibody constant region already present in an expression vector for mammalian cells. Alternatively, the preparation method may involve ligating the chicken leader sequence and variable region sequence present in the cloned cDNA to a sequence encoding a human antibody constant region, followed by ligation into a mammalian cell expression vector. The fragment of the human antibody constant region may be any human antibody heavy chain constant region or any human antibody light chain constant region. Specific examples of the fragment of the human antibody constant region include, for example, Cγ1, Cγ2, Cγ3, or Cγ4 as a human heavy chain. Specific examples of the fragment of the human antibody constant region include, for example, Cλ or Cκ as a human light chain.

[0041] As used herein, the term "humanized antibody" refers to an antibody that is composed of a variable region composed of CDRs from an antibody derived from a non-human animal and a framework region (FR) derived from a human antibody, and a constant region derived from a human antibody. The humanized antibody can be produced by, for example, CDR grafting (Ozaki et al., "Humanized Anti-HM1.24 Antibody Mediates Myeloma Cell Cytotoxicity That Is Enhanced by Cytokine Stimulation of Effector Cells", Blood, 1999 Jun 1;93(11):3922-3930), resurfacing (Roguska et al., "Humanization of murine monoclonal antibodies through variable domain resurfacing", Proc. Natl. Acad. Sci. USA, 1994 Feb 1;91(3):969-973), or FR shuffling (Damschroder et al., "Framework shuffling of antibodies to reduce immunogenicity and manipulate functional and biophysical properties", Mol. Immunol., 2007 Apr;44(11):3049-3060, Epub 2007 Jan 1997). 22), etc. When chicken-derived CDRs are used, the humanized antibody can be prepared, for example, by referring to the method described in Japanese Patent Application Laid-Open No. 2006-241026. In preparing the humanized antibody, amino acid residues in the human FRs may be substituted with corresponding residues from the CDR donor antibody to alter or improve antigen-binding ability. Substitution of amino acid residues in the human FRs can be performed, for example, by methods well known in the art (see, for example, Riechmann et al., "Reshaping human antibodies for therapy", Nature, 1988 Mar. 24; 332 (6162):323-327).

[0042] As used herein, "internalization" or "internalization" means that a cell takes up an antigen on the cell surface into the cell by endocytosis or phagocytosis, thereby taking up a substance bound to the antigen. For example, the anti-CADM1 antibody of the present disclosure has internalization activity, and can thereby internalize a target active ingredient into cells that express CADM1 on their cell surface, thereby producing a desired effect of the target active ingredient in the CADM1-expressing cells.

[0043] As used herein, "antibody drug conjugate" (ADC) refers to an antibody or antigen-binding fragment thereof chemically linked to one or more active ingredients of interest. In the ADC, the active ingredient of interest is operably linked to the antibody or its antigen-binding fragment, for example, via a linker. As used herein, "operably linked" refers to a relationship in which the linked substances are capable of functioning in a predicted manner. Examples of the linker include cleavable and non-cleavable linkers. Examples of the cleavable linker include a linker having a sequence that is cleaved by a protease, a pH-dependent cleavable linker, a disulfide linker, and the like. Examples of the non-cleavable linker include a maleimidomethylcyclohexanecarboxylate (MCC) linker, and the like.

[0044] As used herein, a "bond" may be a covalent bond or a non-covalent bond, for example, an ionic bond, a hydrogen bond, a hydrophobic interaction, or a hydrophilic interaction.

[0045] As used herein, the term "label" refers to a label used to distinguish a molecule or substance of interest from other molecules or substances. Examples of the label include fluorescent labels such as fluorescent dyes or fluorescent substances, such as FITC and rhodamine; enzyme labels such as horseradish peroxidase, β-galactosidase, luciferase, and alkaline phosphatase; chemiluminescent labels; ３ H. １４ C. １５ N. ３５ S.９０ Y. ９９ Tc, １１１ In, １２５ I, １３１ Radioisotopes (RI) such as I; and the like.

[0046] As used herein, "pharmaceutical composition" refers to a chemical compound, composition, drug, or agent capable of inducing a desired therapeutic effect when properly administered to a subject. The pharmaceutical composition may contain one component or multiple components.

[0047] As used herein, the terms "subject" or "administration subject" refer to animals or cells, tissues, or organs derived from animals, and particularly include humans. The term "animal" refers to humans and non-human animals. Examples of non-human animals include mammals such as cows, pigs, sheep, mice, guinea pigs, hamsters, rats, rabbits, horses, cats, dogs, marmosets, monkeys, chimpanzees, dolphins, and sea lions.

[0048] As used herein, "treatment" means therapeutic treatment and / or prophylactic treatment. As used herein, "treatment" means treating, curing, preventing, suppressing, ameliorating, or improving a disease, pathology, or disorder, or halting, inhibiting, reducing, or delaying the progression of a disease, pathology, or disorder. As used herein, "prevention" means reducing the likelihood of developing a disease or pathology, or delaying the onset of a disease or pathology. The "treatment" may be, for example, treatment of a patient who develops a target disease, or treatment of an animal model of the target disease.

[0049] In the present disclosure, a "therapeutically effective amount" refers to the amount of an anti-CADM1 antibody determined to produce a therapeutic response in a subject to which it is administered. The therapeutically effective amount can be readily determined by one skilled in the art.

[0050] As used herein, the term "kit" generally refers to a unit in which the components to be provided (e.g., detection reagents, labels, instructions, etc.) are provided separately in two or more compartments. The kit can be suitably used to provide a composition that is not provided in a mixed state, but is preferably mixed immediately before use, for reasons of stability, etc. The kit preferably includes, for example, instructions or instructions on how to use the components to be provided (e.g., test reagents, etc.), or instructions or instructions describing the processing of the components. As used herein, when the kit is used as a reagent kit, the kit may include instructions, etc., describing how to use the detection reagents, etc.

[0051] As used herein, "instructions" or "instructions" refer to instructions to a physician or other user on how to use the detection reagent, detection kit, or pharmaceutical composition of the present disclosure. The instructions may, for example, describe instructions on how to use the detection reagent, detection kit, or pharmaceutical composition of the present disclosure. The instructions may be prepared in accordance with a format specified by a regulatory agency of the country in which the present disclosure is implemented (e.g., the Ministry of Health, Labor and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, the European Medicines Agency (EMA) in Europe, etc.), and may clearly state that they have been approved by the regulatory agency. The instructions may be a package insert, and are typically provided in paper form, but are not limited thereto. They may also be provided in the form of, for example, an electronic medium (e.g., a website provided on the Internet, email, etc.).

[0052] Sequence information for the proteins described herein or the nucleic acids (e.g., DNA or RNA) encoding them is available from Protein Data Bank, UniProt, GenBank, etc. Furthermore, the nucleic acid sequence of RNA can also be obtained from the corresponding DNA base sequence using appropriate sequence conversion software, etc.

[0053] The present disclosure will be specifically described below using examples. Unless otherwise specified, each disclosure may incorporate the explanations of other disclosures.

[0054] <Pharmaceutical Composition> In another aspect, the present disclosure provides a pharmaceutical composition that can be used for diseases associated with CADM1. The pharmaceutical composition of the present disclosure comprises the anti-CADM1 antibody or antigen-binding fragment thereof of the present disclosure. The pharmaceutical composition of the present disclosure can be used to treat diseases associated with CADM1, as described below. In the following description, unless otherwise specified, the description of the CADM1 antibody can be used to describe the antigen-binding fragment thereof.

[0055] (Anti-CADM1 Antibody and Antigen-Binding Fragment Thereof) In the pharmaceutical composition of the present disclosure, the anti-CADM1 antibody may be any antibody that binds to CADM1. The binding site of the anti-CADM1 antibody is preferably, for example, the extracellular domain of CADM1. Specifically, the anti-CADM1 antibody binds to an epitope containing the amino acid residues at positions 311, 325, and 326 in CADM1 (e.g., SEQ ID NO: 1). The anti-CADM1 antibody or antigen-binding fragment thereof of the present disclosure may recognize, for example, the amino acid residue at position 312 in CADM1. In this case, it can also be said that the anti-CADM1 antibody of the present disclosure binds to an epitope containing the amino acid residues at positions 311, 312, 325, and 326 in CADM1 (e.g., SEQ ID NO: 1). The amino acid residue at position 312 is preferably a tyrosine residue modified with a sulfate group (sulfo group). The anti-CADM1 antibodies of the present disclosure may, for example, bind to a polypeptide consisting of the amino acid sequence from positions 306 to 326 of CADM1 (eg, SEQ ID NO: 1).

[0056] The anti-CADM1 antibody of the present disclosure may be an antibody that binds to wild-type or mutant CADM1. Mutant CADM1 may include those resulting from differences in DNA sequence between individuals, such as single nucleotide polymorphisms (SNPs). The amino acid sequence of the wild-type or mutant CADM1 has, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence set forth in SEQ ID NO: 1 or 2.

[0057] The CADM1 of the present disclosure has reduced binding ability to mutant CADM1 having a mutation in at least one, i.e., one, two, or all (three) amino acid residues selected from the group consisting of positions 311, 325, and 326 in SEQ ID NO: 1, relative to wild-type CADM1 (e.g., SEQ ID NO: 1). The anti-CADM1 antibody of the present disclosure may further have reduced binding ability to mutant CADM1 having a mutation in the amino acid residue at position 312 in SEQ ID NO: 1, relative to wild-type CADM1 (e.g., SEQ ID NO: 1).

[0058] The anti-CADM1 antibodies of the present disclosure may, for example, have reduced binding ability relative to wild-type CADM1 (e.g., SEQ ID NO: 1) to mutant CADM1 having a mutation in at least one, i.e., one, two, or all (three) amino acid residues selected from the group consisting of Y311A, D325A, and Y326A in SEQ ID NO: 1. The anti-CADM1 antibodies of the present disclosure may further have reduced binding ability relative to wild-type CADM1 (e.g., SEQ ID NO: 1) to mutant CADM1 having a mutation in the amino acid residue R312A in SEQ ID NO: 1.

[0059] The anti-CADM1 antibody or antigen-binding fragment thereof of the present disclosure includes, for example, the first antibody described below. The anti-CADM1 antibody or antigen-binding fragment thereof of the present disclosure excludes, for example, the 3E1C antibody described below.

[0060] The anti-CADM1 antibody or antigen-binding fragment thereof of the present disclosure is preferably a monoclonal antibody. The anti-CADM1 monoclonal antibody can be prepared, for example, by the method described above.

[0061] The anti-CADM1 antibody or antigen-binding fragment thereof of the present disclosure is preferably a humanized antibody. The humanized antibody can be produced, for example, by the methods described above.

[0062] The anti-CADM1 antibody or antigen-binding fragment thereof of the present disclosure may have a peptide such as a peptide tag attached to the N-terminus and / or C-terminus of the anti-CADM1 antibody. Examples of the peptide tag include a His tag, a flag™ tag, an HA tag, a T7 tag, a V5 peptide tag, and a Myc tag.

[0063] (First Antibody) The anti-CADM1 antibody (first antibody) of the present disclosure is, for example, an antibody in which heavy chain CDR1-3 and light chain CDR1-3 respectively comprise the amino acid sequences of heavy chain CDR1-3 and light chain CDR1-3 of the 3E1C antibody, 3E1A antibody, 3E1D antibody, 3E1Y antibody, 3E1V antibody, 3E1E antibody, 3E1F antibody, 3E1G antibody, 3E1H antibody, 3E1I antibody, 3E1K antibody, 3E1L antibody, 3E1M antibody, 3E1N antibody, 3E1P antibody, 3E1Q antibody, 3E1R antibody, 3E1S antibody, 3E1W antibody, 3E1T antibody, h3E1A antibody, h3E1F antibody, or h3E1V antibody shown in Tables 1A and 1B below. Note that in each antibody, heavy chain CDR1-2 and light chain CDR1-3 share the same amino acid sequence.

[0064]

[0065]

[0066] Furthermore, the first antibody of the present disclosure is an antibody in which, for example, heavy chain CDR1 to 3 and light chain CDR1 to 3 comprise the amino acid sequences of heavy chain CDR1 to 3 and light chain CDR1 to 3 of the 45TG2 antibody, 45TG3 antibody, 45TG23 antibody, 45TG24 antibody, 45XL7 antibody, 45XL9 antibody, 45XL11 antibody, 45XL14 antibody, 45XL34 antibody, 45XL45 antibody, 43TG47 antibody, 43XL1 antibody, 43XL4 antibody, 43XL17 antibody, 1-1 antibody, 1-2 antibody, 1-3 antibody, 1-4 antibody, 2-1 antibody, 2-2 antibody, 2-3 antibody, 2-4 antibody, 3-1 antibody, 3-2 antibody, 3-3 antibody, 3-4 antibody, 4-1 antibody, 4-2 antibody, 4-3 antibody, or 4-4 antibody, respectively, shown in Tables 2A to 2C below.

[0067]

[0068]

[0069]

[0070] The first antibody of the present disclosure is, for example, an antibody whose heavy chain variable region and light chain variable region comprise the amino acid sequences of the heavy chain variable region and light chain variable region of the 3E1C antibody, 3E1A antibody, 3E1D antibody, 3E1Y antibody, 3E1V antibody, 3E1E antibody, 3E1F antibody, 3E1G antibody, 3E1H antibody, 3E1I antibody, 3E1K antibody, 3E1L antibody, 3E1M antibody, 3E1N antibody, 3E1P antibody, 3E1Q antibody, 3E1R antibody, 3E1S antibody, 3E1W antibody, 3E1T antibody, h3E1A antibody, h3E1F antibody, or h3E1V antibody shown in Tables 1A to 1B. Note that the light chain variable region of each antibody has a common amino acid sequence.

[0071] Furthermore, the first antibody of the present disclosure is an antibody whose heavy chain variable region and light chain variable region comprise the amino acid sequences of the heavy chain variable region and light chain variable region of the 45TG2 antibody, 45TG3 antibody, 45TG23 antibody, 45TG24 antibody, 45XL7 antibody, 45XL9 antibody, 45XL11 antibody, 45XL14 antibody, 45XL34 antibody, 45XL45 antibody, 43TG47 antibody, 43XL1 antibody, 43XL4 antibody, 43XL17 antibody, 1-1 antibody, 1-2 antibody, 1-3 antibody, 1-4 antibody, 2-1 antibody, 2-2 antibody, 2-3 antibody, 2-4 antibody, 3-1 antibody, 3-2 antibody, 3-3 antibody, 3-4 antibody, 4-1 antibody, 4-2 antibody, 4-3 antibody, 4-4 antibody, or H3LA2 antibody, respectively, shown in Tables 2A to 2C.

[0072] In the first antibody of the present disclosure, the above-listed amino acid sequences may be (i) the above amino acid sequences in which one or more amino acids have been deleted, substituted, inserted, or added, (ii) amino acid sequences that are 80% or more identical to the above amino acid sequences, and / or (iii) amino acid sequences encoded by a polynucleotide that specifically hybridizes under stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to a nucleotide sequence encoding the above amino acid sequence, so long as the anti-CADM1 antibody has binding affinity to wild-type CADM1 (e.g., SEQ ID NO: 1 or 2). The above (i) to (iii) also apply to amino acid sequences listed in the sequence listing. In this case, when the sequence is a nucleic acid, the "amino acid sequence" in the above explanation can be read as "nucleotide sequence," and the explanation therefor can be used.

[0073] In (i) above, "one or several" may be, for example, 1 to 24, 1 to 20, 1 to 15, 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1, or may be any of these values ​​or less. It is known that polypeptides in which one or more amino acid residues have been deleted, added, inserted, or substituted with other amino acids (hereinafter collectively referred to as "mutations") maintain their biological activity (Mark et al., Proc Natl Acad Sci U.S.A. 1984 Sep;81(18):5662-5666; Zoller et al., Nucleic Acids Res. 1982 Oct 25;10(20):6487-6500; Wang et al., Science. 1984 Jun 29;224(4656):1431-1433). Mutated antibodies can be produced, for example, by site-directed mutagenesis, random mutagenesis, or biopanning using an antibody phage library. Site-directed mutagenesis can be performed using, for example, the KOD-Plus-Mutagenesis Kit (TOYOBO CO., LTD.). Selection of an antibody having activity similar to that of the wild-type antibody from among the mutant antibodies into which the mutations have been introduced can be achieved by various characterization methods, such as FACS analysis and ELISA. In (i), the mutations are present, for example, in the CDR or FR of the amino acid sequence, and are preferably present in the FR. When the mutations are present in the CDR, the "one or several" refers to, for example, 3, 2, or 1 in total, which is the total number of deletions, etc., in the amino acid sequence of the entire CDR.

[0074] In (i), the mutation is preferably a substitution, insertion, deletion, or addition to at least one of the N-terminus and C-terminus of the CDR. The substitution is preferably a conservative substitution (the same applies hereinafter). The "conservative substitution" refers to substituting one or several amino acids with other amino acids and / or amino acid derivatives so as not to substantially alter the function of the protein. The "substituting amino acid" and the "substituted amino acid" preferably have similar properties and / or functions. Specifically, they preferably have similar chemical properties, such as hydrophobicity and hydrophilicity index (hydropathy), polarity, and charge, or physical properties, such as secondary structure. Amino acids or amino acid derivatives with similar properties and / or functions are known in the art, for example. Specific examples of nonpolar amino acids (hydrophobic amino acids) include alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine; polar amino acids (neutral amino acids) include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine; positively charged amino acids (basic amino acids) include arginine, histidine, and lysine; and negatively charged amino acids (acidic amino acids) include aspartic acid and glutamic acid.

[0075] In (ii) above, the "identity" may be, for example, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or 100%, or may be within a range of any two of these values.

[0076] In (iii) above, "stringent conditions" may be, for example, low stringency conditions, moderate stringency conditions, or high stringency conditions. "Low stringency conditions" are, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 32°C. "Medium stringency conditions" are, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 42°C. "High stringency conditions" are, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 50°C. Those skilled in the art can set the degree of stringency by appropriately selecting conditions such as temperature, salt concentration, probe concentration and length, ionic strength, and time. The term "stringent conditions" is defined, for example, as described in Sambrook et al., eds., Molecular Cloning: A Laboratory Manual 2nd Edition. nd The conditions described in "Cold Spring Harbor Laboratory Press (1989)" can also be used.

[0077] In the above (iii), the "hybridizing polynucleotide" is, for example, a polynucleotide that is completely or partially complementary to the polynucleotide encoding the amino acid sequence. The hybridization can be detected, for example, by various hybridization assays. The hybridization assay is not particularly limited, and examples thereof include the hybridization assays described in "Molecular Cloning: A Laboratory Manual 2nd Edition" edited by Sambrook et al. nd Alternatively, the method described in "Cold Spring Harbor Laboratory Press (1989)" or the like can be employed.

[0078] The first antibody having any of the amino acid sequences (i) to (iii) preferably comprises, for example, a part, preferably the entire amino acid sequence of heavy chain CDR1 to 3 contained in the corresponding heavy chain variable region. Also, the first antibody having any of the amino acid sequences (i) to (iii) preferably comprises, for example, a part, preferably the entire amino acid sequence of light chain CDR1 to 3 contained in the corresponding light chain variable region.

[0079] The first antibody of the present disclosure preferably has reduced non-specific binding compared to, for example, the 3E1 antibody. The reduced non-specific binding can be evaluated using binding to BSA as an index in accordance with Example 1 (1-5) described below. Specifically, when evaluating the binding to 20 μg / ml of a target antibody or the 3E1 antibody, the measured value of the target antibody can be evaluated as having reduced non-specific binding if, for example, the measured value of the 3E1 antibody is 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less, based on the measurement of the 3E1 antibody (100%).

[0080] The first antibody of the present disclosure preferably exhibits suppressed antibody degradation compared to, for example, the 3E1 antibody. The suppression of antibody degradation can be evaluated using degradation products resulting from degradation of the target antibody as an indicator, in accordance with Example 1 (1-3) described below. Specifically, when 1 μg of the target antibody or the 3E1 antibody is electrophoresed, the amount of degradation products of the target antibody is, for example, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of the amount of degradation products of the 3E1 antibody (100%). The target antibody can be evaluated as having suppressed antibody degradation.

[0081] (Second Antibody) The anti-CADM1 antibody (second antibody) of the present disclosure is, for example, an antibody in which heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3 comprise the amino acid sequences of heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3 of the 9D2 antibody, H1L1 antibody, H1L2 antibody, H1L3 antibody, H1LA1 antibody, H1LA2 antibody, H2L1 antibody, H2L2 antibody, H2L3 antibody, H2LA1 antibody, H2LA2 antibody, H3L1 antibody, H3L2 antibody, H3LA1 antibody, or H3LA2 antibody, respectively, shown in Tables 3A and 3B below.

[0082]

[0083]

[0084] The second antibody of the present disclosure is, for example, an antibody whose heavy chain variable region and light chain variable region comprise the amino acid sequences of the heavy chain variable region and light chain variable region of the 9D2 antibody, H1L1 antibody, H1L2 antibody, H1L3 antibody, H1LA1 antibody, H1LA2 antibody, H2L1 antibody, H2L2 antibody, H2L3 antibody, H2LA1 antibody, H2LA2 antibody, H3L1 antibody, H3L2 antibody, H3LA1 antibody, or H3LA2 antibody shown in Tables 3A and 3B, respectively.

[0085] In the second antibody of the present disclosure, the above-listed amino acid sequences may be (i) the above amino acid sequences in which one or more amino acids have been deleted, substituted, inserted, or added, (ii) an amino acid sequence that is 80% or more identical to the above amino acid sequence, and / or (iii) an amino acid sequence encoded by a polynucleotide that specifically hybridizes under stringent conditions to a polynucleotide consisting of a nucleotide sequence complementary to a nucleotide sequence encoding the above amino acid sequence, so long as the anti-CADM1 antibody has binding affinity to wild-type CADM1 (e.g., SEQ ID NO: 1 or 2). The above (i) to (iii) also apply to amino acid sequences listed in the Sequence Listing. In this case, if the sequence is a nucleic acid, the "amino acid sequence" in the above explanation can be read as "nucleotide sequence," and the explanation therefor can be used. The explanations for (i) to (iii) for the first antibody can be used for the above (i) to (iii).

[0086] The second antibody having any one of the amino acid sequences (i) to (iii) preferably comprises, for example, a part, preferably the entire amino acid sequence of heavy chain CDR1 to 3 contained in the corresponding heavy chain variable region. Also, the second antibody having any one of the amino acid sequences (i) to (iii) preferably comprises, for example, a part, preferably the entire amino acid sequence of light chain CDR1 to 3 contained in the corresponding light chain variable region.

[0087] The second antibody of the present disclosure is, for example, an IgM antibody, preferably a humanized IgM antibody.

[0088] The second antibody of the present disclosure can promote the internalization of other anti-CADM1 antibodies, such as the first antibody, and is therefore expected to enhance the activity of these antibodies, for example, when used in combination with the first antibody.

[0089] The anti-CADM1 antibody of the present disclosure is preferably a monoclonal antibody, which can be prepared, for example, by the method described above.

[0090] The anti-CADM1 antibody of the present disclosure is preferably a humanized antibody, more preferably a humanized IgG antibody. The humanized antibody can be prepared, for example, by the method described above.

[0091] The anti-CADM1 antibody of the present disclosure may have a peptide such as a peptide tag attached to its N-terminus and / or C-terminus. Examples of the peptide tag include a His tag, a flag™ tag, an HA tag, a T7 tag, a V5 peptide tag, and a Myc tag.

[0092] The anti-CADM1 antibody of the present disclosure can be produced, for example, by immunizing a mammal or bird with CADM1 or a peptide thereof and selecting an antibody that binds to CADM1 from the resulting antibody population. The selection can be performed, for example, using a protein having the amino acid sequence shown in SEQ ID NO: 1.

[0093] The anti-CADM1 antibody of the present disclosure is capable of binding to CADM1. Thus, the antibody or antigen-binding fragment thereof of the present disclosure can be suitably used for, for example, detecting CADM1, treating diseases associated with CADM1, such as cancers such as mesothelioma, gastrointestinal stromal tumor, lung adenocarcinoma, endometrial adenocarcinoma, osteosarcoma, and adult T-cell leukemia; atopic dermatitis; suppressing peripheral neuralgia (e.g., traumatic pain, chemical traumatic pain, inflammatory pain, cancer pain, chemotherapy-induced peripheral neuropathy, neuropathic pain, and postherpetic neuralgia); suppressing pruritus; and the like.

[0094] Furthermore, the anti-CADM1 antibody of the present disclosure can be delivered intracranially via the nasal route. Therefore, the anti-CADM1 antibody of the present disclosure is expected to be suitable for treating brain diseases, such as brain tumors, Alzheimer's disease, stroke, epilepsy, Parkinson's disease, headache, and demyelinating neurological diseases, when used, for example, as an antibody in an antibody-drug conjugate described below.

[0095] Furthermore, the anti-CADM1 antibody of the present disclosure can be delivered into mast cells, peripheral nerves, or osteoblasts. Therefore, when used as an antibody in an antibody-drug conjugate described below, the anti-CADM1 antibody of the present disclosure is expected to be suitable for the treatment of mast cell-associated allergic diseases, peripheral nerve-associated neurological diseases such as peripheral neuralgia, bone diseases such as osteoporosis, intractable fractures, and intraosseous pseudoarthrosis, type 1 diabetes, and the like.

[0096] The anti-CADM1 antibody of the present disclosure may be in the form of an antibody-drug conjugate. In this case, the pharmaceutical composition of the present disclosure contains, for example, the anti-CADM1 antibody or antigen-binding fragment thereof of the present disclosure and a target active ingredient (drug).

[0097] The target active ingredient can be any ingredient. Examples of the target active ingredient include cytotoxic agents, anesthetics, diabetic neuropathy treatment drugs, Alzheimer's disease treatment drugs, bone formation promoting drugs, anticancer drugs, contrast agents, proteins such as antibodies, growth factors, siRNA, antisense nucleic acids, ribozymes, etc. Examples of the cytotoxic agent include alkylating agents, tumor necrosis factor inhibitors, intercalators, microtubule inhibitors, kinase inhibitors, proteasome inhibitors, topoisomerase inhibitors, etc. Specific examples of the cytotoxic agent include auristatins (e.g., monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF)), maytansinoids (e.g., DM1 or DM4), calicheamicin, duocarmycin, pyrrolobenzodiazepines (PBDs), etc. Examples of the anesthetic include local anesthetics such as procaine. Examples of the bone formation promoting agent include estrone, etc. Examples of the diabetic neuropathy therapeutic agent include epalrestat, etc. Examples of the Alzheimer's disease therapeutic agent include β-secretase inhibitors, etc.

[0098] In the antibody-drug conjugate, the antibody or antigen-binding fragment thereof and the active ingredient of interest may be directly bound (linked) or indirectly bound (linked) via a linker. In the latter case, the linker may be a cleavable linker that is cleaved by an enzyme such as a protease or peptidase, pH, or the like, or a non-cleavable linker. The cleavable linker is designed, for example, to have a cleavage site that is not cleaved by enzymes present in the blood but is cleaved only by enzymes present within cells. Specific examples of the cleavable linker include valine-citrulline (Val-Cit), valine-alanine (Val-Ala), and alanine-alanine-asparagine (Ala-Ala-Asn).

[0099] In the antibody-drug conjugate of the present disclosure, the linker and the component of interest (drug) are represented, for example, by the following formula (1):

[0100] The pharmaceutical composition of the present disclosure can be administered to, for example, humans or non-human animals (e.g., mammals such as cows, pigs, sheep, mice, guinea pigs, hamsters, rats, rabbits, horses, cats, dogs, marmosets, monkeys, chimpanzees, dolphins, and sea lions).

[0101] The pharmaceutical composition of the present disclosure is preferably administered via an effective route for treatment, such as intravenous, subcutaneous, intramuscular, intraperitoneal, epidural, or oral administration. The pharmaceutical composition may be administered in the form of, for example, an injection, infusion, capsule, tablet, or granule. When administering the antibody, it is effective to administer the pharmaceutical composition as an injection. The aqueous solution for injection may be stored, for example, in a vial or stainless steel container. The aqueous solution for injection may also contain, for example, physiological saline, sugar (e.g., trehalose), NaCl, or NaOH. The pharmaceutical composition may also contain, for example, an effective amount of a buffer (e.g., phosphate buffer), a pH adjuster, a stabilizer, or the like.

[0102] The dosage of the pharmaceutical composition of the present disclosure is, for example, a therapeutically effective amount, specifically, 0.01 to 200 mg / kg body weight per administration. The administration interval is not particularly limited, and may be, for example, once or twice every 1 to 28 days. The dosage, administration interval, and administration method may be appropriately selected depending on, for example, the age, body weight, symptoms, target organ, etc. of the subject.

[0103] In the pharmaceutical composition of the present disclosure, the anti-CADM1 antibody can be obtained, for example, by immunizing a mammal or bird with CADM1 or a peptide thereof. Alternatively, the anti-CADM1 antibody may be prepared, for example, by selecting, from the resulting antibody population, an antibody that specifically binds to an epitope containing the amino acid residue at the predetermined position. The selection can be performed, for example, using the mutant CADM1 described above.

[0104] The anti-CADM1 antibody may be produced by genetic engineering techniques, in which case the anti-CADM1 antibody can be produced by expressing the anti-CADM1 antibody in a transformant using a nucleic acid encoding the anti-CADM1 antibody.

[0105] (Nucleic Acid) The nucleic acid can be obtained, for example, by the following method. First, RNA is extracted from a hybridoma producing the anti-CADM1 antibody of the present disclosure, and then cDNA is synthesized using reverse transcriptase. The obtained cDNA is amplified using primers for sequences conserved in the variable regions of the heavy chain gene and the light chain gene, and DNA sequence information can be determined from the obtained amplified fragment. Alternatively, DNA encoding the anti-CADM1 antibody can be obtained from the obtained base sequence information by chemically synthesizing the sequence of the variable region or a portion thereof and linking it to a sequence containing the constant region.

[0106] The nucleic acids of the present disclosure may be operably linked to a vector, such as, for example, a plasmid vector.

[0107] (Transformant) The transformant can be produced, for example, by introducing the nucleic acid into a host cell.

[0108] Examples of host cells for the transformant include prokaryotic cells such as Escherichia coli and Bacillus subtilis, and eukaryotic cells, preferably eukaryotic cells, and more preferably mammalian-derived cells, such as Chinese hamster ovary cells (CHO cells), monkey cells COS-7, human embryonic kidney cells (e.g., HEK293 cells), myeloma, BHK, HeLa, Vero, 293, NS0, Namalwa, and YB2 / 0.

[0109] The nucleic acid may be a vector containing the nucleic acid. The vector can be appropriately selected depending on, for example, the type of transformant. Examples of the vector that can be expressed in mammalian cells include plasmid vectors such as pcDNA3.1 (manufactured by Invitrogen), pConPlus, pcDM8, pcDNA I / Amp, pcDNA3.1, pREP4, pA1-11, and pcDNA3.1-V5 / His-TOPO; viral vectors such as pDON-AI DNA (manufactured by Takara Bio Inc.); and bacteriophages such as λ phage. The vector may be an expression vector or may be circular.

[0110] The nucleic acid or vector can be introduced into host cells by, for example, the calcium phosphate method, lipofection, electroporation, adenovirus-based methods, retrovirus-based methods, or microinjection (New Genetic Engineering Handbook, 4th revised edition, Yodosha (2003): 152-179). Antibodies can be produced using the cells by, for example, the method described in Protein Experiment Handbook, Yodosha (2003): 128-142).

[0111] To produce the anti-CADM1 antibody, for example, a host cell (transformant) containing the nucleic acid or vector may be grown. The growth may include, for example, culturing.

[0112] The anti-CADM1 antibody can be recovered and purified (isolated) from the culture medium of the transformant, for example. The purification can be performed using a method typically used for separating and purifying antibodies and the like. For example, the antibody can be separated and purified using a chromatography column such as affinity chromatography, a filter, ultrafiltration, salting out, dialysis, SDS-polyacrylamide gel electrophoresis, isoelectric focusing, or the like, selected as appropriate, either alone or in combination.

[0113] The CADM1 may be added with a target active ingredient via, for example, a linker. The addition can be set, for example, depending on the linker and the target ingredient.

[0114] The anti-CADM1 antibody of the present disclosure is capable of binding to CADM1. Thus, the anti-CADM1 antibody of the present disclosure can be suitably used for the treatment of diseases associated with CADM1, such as cancers such as mesothelioma, gastrointestinal stromal tumor, lung adenocarcinoma, endometrial adenocarcinoma, osteosarcoma, and adult T-cell leukemia; atopic dermatitis; suppression of peripheral neuralgia (e.g., traumatic pain, chemical traumatic pain, inflammatory pain, cancer pain, chemotherapy-induced peripheral neuropathy, neuropathic pain, postherpetic neuralgia, etc.); suppression of pruritus; and the like.

[0115] Furthermore, the anti-CADM1 antibody of the present disclosure can be delivered intracranially via the nasal route. Therefore, the anti-CADM1 antibody of the present disclosure is expected to be suitable for treating brain diseases, such as brain tumors, Alzheimer's disease, stroke, epilepsy, Parkinson's disease, headache, and demyelinating neurological diseases, when used, for example, as an antibody in an antibody-drug conjugate described below.

[0116] Furthermore, the anti-CADM1 antibody of the present disclosure can be delivered into mast cells, peripheral nerves, or osteoblasts. Therefore, the anti-CADM1 antibody or antigen-binding fragment thereof of the present disclosure, when used as an antibody in the antibody-drug conjugate, is expected to be suitable for the treatment of mast cell-associated allergic diseases, peripheral nerve-associated neurological diseases such as peripheral neuralgia, bone diseases such as osteoporosis, intractable fractures, and intraosseous pseudoarthrosis, and type 1 diabetes.

[0117] The pharmaceutical compositions of the present disclosure may further be accompanied by instructions or directions.

[0118] <Treatment Method> In another aspect, the present disclosure provides a treatment method that can be performed for a disease associated with CADM1. The treatment method of the present disclosure comprises a step of using an anti-CADM1 antibody or antigen-binding fragment thereof of the present disclosure. The treatment method of the present disclosure comprises, for example, an administration step of administering an anti-CADM1 antibody or antigen-binding fragment thereof to a subject. The treatment method of the present disclosure can be used to treat a disease associated with CADM1.

[0119] In the treatment methods of the present disclosure, the use may be in vitro or in vivo.

[0120] <Delivery Compositions and Delivery Methods> In another aspect, the present disclosure provides compositions and delivery methods that can be used for nasal intracranial delivery, intraosseous or peripheral nerve delivery, and / or delivery to mast cells or osteoblasts. A composition (first composition) for use in intracranial delivery by nasal administration of the present disclosure comprises an anti-CADM1 antibody or antigen-binding fragment thereof and a drug. A composition (second composition) for use in intraosseous or peripheral nerve delivery of the present disclosure comprises an anti-CADM1 antibody or antigen-binding fragment thereof and a drug. Furthermore, a composition (third composition) for use in delivery to mast cells or osteoblasts of the present disclosure comprises an anti-CADM1 antibody or antigen-binding fragment thereof and a drug.

[0121] The drug delivery method of the present disclosure includes the step of intranasally administering a first composition of the present disclosure to a subject to deliver the drug intracranially. The drug delivery method of the present disclosure includes the step of administering a second composition of the present disclosure to a subject to deliver the drug intraosseously or to a peripheral nerve. The drug delivery method of the present disclosure includes the step of administering a third composition of the present disclosure to a subject to deliver the drug to mast cells or osteoblasts. The compositions and delivery methods of the present disclosure can be referenced from the description of the pharmaceutical compositions of the present disclosure.

[0122] <Use> The present disclosure relates to a pharmaceutical composition of the present disclosure for use in a method for treating a CADM1-associated disease. The present disclosure relates to use of a pharmaceutical composition of the present disclosure for use in treating a CADM1-associated disease for the manufacture of a pharmaceutical composition for use in treating a CADM1-associated disease. The present disclosure relates to a composition of the present disclosure for use in intracranial delivery by nasal administration for use in a method of intracranial delivery of a drug by nasal administration. The present disclosure relates to use of a composition of the present disclosure for use in intracranial delivery by nasal administration for the manufacture of a composition for use in intracranial delivery of a drug by nasal administration. The present disclosure relates to a composition of the present disclosure for use in intraosseous or peripheral nerve delivery for use in a method of intraosseous or peripheral nerve delivery. The present disclosure relates to use of a composition of the present disclosure for use in intraosseous or peripheral nerve delivery for the manufacture of a composition for use in intraosseous or peripheral nerve delivery. The present disclosure is a composition for use in a method of delivery to mast cells or osteoblasts, and the present disclosure is the use of a composition for use in delivery to mast cells or osteoblasts, and the present disclosure is the use of a composition for use in delivery to mast cells or osteoblasts, for the manufacture of a composition for use in delivery to mast cells or osteoblasts.

[0123] Next, examples of the present disclosure will be described. However, the present disclosure is not limited by the following examples. Commercially available reagents were used according to their protocols unless otherwise specified. Note that "mol / l" may also be abbreviated as "M". [Example 1]

[0124] (1) Mouse Chimeric 3E1 Antibody and Its Mutants Mouse chimeric 3E1 antibody and its mutants were produced, and the affinity and degradability of the antibodies were examined.

[0125] (1-1) Preparation of Mouse Chimeric 3E1 Antibody First, a chicken anti-CADM1 antibody (chicken 3E1 antibody) was prepared. Chickens were used as immunized animals. The antigen used was a recombinant protein of the full-length extracellular domain of mouse CADM1 (Furuno T. et al., Journal of Immunology, 2005). After immunizing the chickens with the antigen, antibody-producing cells were collected from the spleen and fused with the chicken B cell line MuH1. Hybridoma clones producing anti-CADM1 antibodies were selected by ELISA based on their reactivity to CADM1-Fc (Koma Y. et al., Oncogene, 2004), which is a mouse IgG Fc region added to the C-terminus of the extracellular domain of mouse CADM1.

[0126] The nucleic acid sequence encoding the obtained chicken anti-CADM1 antibody was decoded from the hybridoma producing the antibody, and the amino acid sequence was identified from the nucleic acid sequence. As a result, the chicken anti-CADM1 antibody was found to comprise a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 6 and a light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 10. Heavy chain CDRs 1 to 3 (SEQ ID NOs: 3 to 5) and light chain CDRs 1 to 3 (SEQ ID NOs: 7 to 9) of the chicken anti-CADM1 antibody were identified according to the Kabat numbering system (the same applies hereinafter). 3E1 antibody Heavy chain CDR1: SNAMG (SEQ ID NO: 3) Heavy chain CDR2: GIGNTGRYTGYGPAVKG (SEQ ID NO: 4) Heavy chain CDR3: SASGSGYGCAGWIDA (SEQ ID NO: 5) Heavy chain variable region: AVTLDESGGGLQTPGGALSLVCKASGFTFSSNAMGWVRQAPGKGLEFVAGIGNTGRYTGYGPAVKGRATISRDNGQSTVRLQLNNLRAEDTAIYYCAKSASGSGYGCAGWIDAWGHGTEVIVSS (SEQ ID NO: 6) Light chain CDR1: SGGSYIYG (SEQ ID NO: 7) Light chain CDR2: SNDKRPS (SEQ ID NO: 8) Light chain CDR3: GNEDNSIDSAI (SEQ ID NO: 9) Light chain variable region: ALTQQPASVSANPGETVKITCSGGSYIYGWYQQKSPGSAPVTVIYSNDKRPSNIPSRFSGSTSGSTGTLTITGVQADDEAVYFCGNEDNSIDSAIFGAGTTLTVL (SEQ ID NO: 10)

[0127] Genes encoding the heavy and light chains of the 3E1 antibody (chicken type) from the hybridoma were inserted into an expression vector. Next, the heavy and light chain variable regions were amplified by PCR using the expression vector containing the chicken type 3E1 antibody gene as a template. The resulting amplified fragments of the heavy and light chain variable regions were inserted by homologous recombination into expression vectors containing the constant regions of mouse IgG1 (heavy chain) and mouse κ chain (light chain), respectively, using a cloning kit (Seamless Cloning and Assembly Enzyme Mix, Thermo Fisher Scientific, Cat. No.: A14606). The resulting expression vector encoding the mouse chimeric antibody was subjected to sequence analysis to confirm that nucleic acids encoding the mouse chimeric antibody had been introduced.

[0128] (1-2) Construction of Mutants of Mouse Chimeric 3E1 Antibody Mutants of the mouse chimeric 3E1 parent antibody were prepared. Specifically, Cys present in CDR3 of the heavy chain variable region of the mouse chimeric 3E1 antibody (3E1 parent antibody) prepared in Example 1 (1-1) above was substituted with another amino acid. For the substitution, primers were designed to introduce "NNN" (where N is A, T, G, or C) into the base sequence site of Cys, and random mutagenesis was performed using a KOD-Plus-Mutagenesis Kit (TOYOBO, SMK-101). The sequences of the primers are shown in Table 4 below. After the mutagenesis, the substituted amino acids were identified by sequence analysis. Table 1 above shows the amino acid sequences of the heavy chain variable regions of 3E1 and its mutants.

[0129]

[0130] The heavy chain expression vectors containing each mutation were transiently expressed together with the light chain expression vector for the 3E1 parent antibody using the Expi293 Expression System (Thermo Fisher Scientific, A14635) in a 25 ml volume according to the protocol. The amino acid sequence of the 3E1 light chain variable region (SEQ ID NO: 10) is shown in Table 5 below. After expression, each culture supernatant was purified using Ab-Capcher Extra (Protenova, P-003-200) to obtain purified antibodies of each mouse chimeric 3E1 mutant. In the following figures, 3E1 and 3E1 mutants may be shown only with the amino acid residue before substitution (C) and the amino acid residue after substitution.

[0131]

[0132] (1-3) Study on Degradation of Mouse Chimeric 3E1 Mutants Next, SDS-PAGE was performed on each of the purified antibodies. Specifically, the 3E1 parent antibody obtained in Example 1 (1-1) and each of the purified antibodies obtained in Example 1 (1-2) were adjusted to a final application amount of 1 μg and heated at 70°C for 10 minutes. After the heat treatment, each purified antibody was applied to NuPAGE™ 4 to 12% Bis-Tris, 1.0-1.5 mm (Thermo Fisher Scientific, NP0329BOX) and electrophoresed at a constant voltage of 114 V for 90 minutes. The electrophoresis buffer used was NuPAGE™ MES SDS Running Buffer (Thermo Fisher Scientific, NP0002). The gel was stained using Quick-CBB PLUS (Fujifilm Wako Pure Chemical Industries, Ltd., #178-00551). The results are shown in Figure 1.

[0133] Figure 1 shows the results of SDS-PAGE. In Figure 1, the vertical axis represents molecular weight, and the horizontal axis represents the substituted amino acid for each purified antibody. As shown in Figure 1, for the 3E1 parent antibody, bands were observed around 150 kDa and between 100 and 150 kDa, whereas for each purified mutant antibody, a band was observed only around 150 kDa. These results demonstrate that antibody degradation is suppressed by substituting a cysteine ​​present in CDR3 of the 3E1 heavy chain variable region with another amino acid.

[0134] (1-4) Study of the Affinity of Each Purified Antibody of Mouse Chimeric 3E1 Mutants The affinity of each purified antibody of mouse chimeric 3E1 mutants was studied. Specifically, the affinity was measured using single-cycle kinetics. The 3E1 parent antibody obtained in Example 1 (1-1) and each purified antibody of mouse chimeric 3E1 mutant obtained in Example 1 (1-2) were adjusted to 40 nmol / L using running buffer HBS-EP+ buffer (Cytiva, BR100669) to obtain a ligand solution. The antigen CADM1-NTF-HisTag was diluted with running buffer to prepare five 3-fold dilutions starting from 10 nmol / L to obtain an analyte solution. The running buffer was also used as a blank solution. Next, the ligand solution was added to Flow Cell 2 at a flow rate of 10 μl / min for 60 seconds. After the addition, the blank solution or the analyte solution was added to Flow Cells 1 and 2 at a flow rate of 30 μl / min for 120 seconds. The dissociation time was 1200 seconds. Analysis was performed using Biacore™ Insight Evaluation Software (Cytiva), and binding parameters were calculated using a 1:1 binding model. The results are shown in Table 6 below. As shown in Table 6 below, each purified antibody of the mouse chimeric 3E1 mutant was found to exhibit high affinity, similar to the 3E1 parent antibody.

[0135]

[0136] (1-5) Examination of the reactivity of each purified antibody of the mouse chimeric 3E1 mutant by ELISA The reactivity of each purified antibody of the mouse chimeric 3E1 mutant was examined by ELISA. Specifically, 5 μg / ml BSA (T9310A, manufactured by Takara Bio Inc.) was immobilized on an immunoplate (442404, manufactured by Thermo Fisher Scientific) at 4°C overnight. After immobilization, the immobilized solution was removed and the plate was washed with PBS-T (405TS, manufactured by BioTek). After washing, 20% blocking solution (Blocking One, manufactured by Nacalai Tesque Inc., 03953-95) was added and incubated at 37°C for 1 hour. After incubation, the plate was washed with PBS-T. After washing, the primary reaction was carried out at 37°C for 1 hour using the purified 3E1 parent antibody obtained in Example 1 (1-1) and the mouse chimeric 3E1 mutant antibody obtained in Example 1 (1-2). The antibody concentrations were 100 μg / ml and 8 5-fold dilutions (0.0064, 0.032, 0.16, 0.8, 4, 20, or 100 μg / ml). After the primary reaction, the cells were washed three times with PBS-T, and then a secondary reaction was carried out at 37°C for 1 hour using a secondary antibody (HRP-anti-mouse IgG, Seracare, 5450-0011) diluted to 0.5 μg / ml. After the secondary reaction, the cells were washed three times with PBS-T. After washing, TMB (KPL, 5120-0076) was added, and color development was carried out at room temperature (hereinafter, approximately 25°C) for 20 minutes. After the color development, TMB stop solution (KPL, 5150-0020) was added to stop the reaction. Then, absorbance (450 nm / 650 nm) was detected using a plate reader (Cytation, BioTek). The results are shown in Figure 2.

[0137] Figure 2 is a graph showing the results of ELISA. In Figure 2, the vertical axis represents absorbance, and the horizontal axis represents the concentration (μg / ml) of the primary antibody. As shown in Figure 2, each purified antibody of the 3E1 mutants showed reduced nonspecific binding compared to the 3E1 parent antibody.

[0138] (2) Humanized 3E1 (h3E1C) and its mutants Humanized 3E1C antibody and its mutants were prepared, and the affinity and degradability of the antibodies were examined.

[0139] (2-1) Design of Humanized Anti-CADM1 Antibody The humanized anti-CADM1 antibody (humanized 3E1C) was constructed primarily by CDR grafting. Specifically, the following steps 1) to 4) were performed. 1) CDR sequences were selected from the base sequence of the anti-CADM1 chicken antibody, and these CDRs were grafted onto the human framework sequence. 2) The sequence of the chicken anti-CADM1 antibody was compared with the human framework sequence, and the amino acid sequence differences in the Vernier Zone were identified. 3) Codons using mixed bases were designed so that the different sequence positions could be translated into either human-derived or chicken-derived amino acids (in some cases, amino acids different from both may appear). 4) The designed VH and VL base sequences were combined to form an scFv (single chain Fv) construct.

[0140] (2-2) Preparation of a humanized anti-scFv library The designed sequence was synthesized, amplified by PCR, and inserted into the pPDS(hCκ) phagemid vector (Yamanaka IH. et al., Journal of Biochemistry, 1995). The gene was then introduced into XL1-Blue Escherichia coli (Stratagene, 200228). Helper phage was added to the bacterial culture, followed by IPTG (100 μg / ml) to express the phage scFv antibody. The next day, PEG precipitation was performed, and the precipitate was dissolved in PBS (phosphate-buffered saline) to obtain a primary scFv antibody library.

[0141] (2-3) Panning of the Primary scFv Antibody Library Specific binding to the antigen, washing (removal of nonspecific antibodies), and recovery of specific antibodies were performed as follows. First, 1 μg / ml of CADM1-Fc fusion protein was immobilized on an immunoplate (Maxisorp, Nunc), and then the scFv antibody library was added to allow specific binding to the antigen. The wells were washed with PBS-T to remove nonspecific antibodies, followed by addition of XL1-Blue and infection with phage (recovery of specific antibodies and reinfection of E. coli). IPTG was added to induce expression of phage scFv antibodies. The following day, PEG precipitation was performed, and the precipitate was dissolved in PBS (panning scFv antibody library). The above panning procedure was repeated 3 to 5 times.

[0142] (2-4) Phage scFv Antibody Expression and Screening (ELISA) The panning scFv library was mixed with XL1-Blue and plated on a 2xYT plate to form colonies and isolate the clones. The colonized clones were cultured in 2xYT medium, and helper phage and IPTG were added to express the scFv antibodies. After expression, the culture supernatant was collected and subjected to ELISA screening. The ELISA was performed using an immunoplate (Maxisorp, Nunc) coated with CADM1-Fc or BSA, and a secondary antibody (HRP-anti-human-kappa, Thermo Fisher Scientific). Clones that showed high reactivity with CADM1-Fc and no nonspecific reactivity with BSA were selected.

[0143] (2-5) Preparation of Bivalent Antibodies The selected clones and clones (Wt) in which all framework amino acids were replaced with human amino acids were recombined into human IgG4 / κ or IgG1 / κ as follows. First, for Wt, the heavy and light chain variable regions were amplified by PCR using fully synthetic DNA as a template. For the selected clones, the heavy and light chain variable regions were amplified by PCR using an scFv expression plasmid as a template. The resulting PCR products were then inserted by homologous recombination into pre-constructed expression vectors (pcDNA3.4, Thermo Fisher Scientific) containing the constant regions of human IgG4 or IgG1 (heavy chain) and human κ chain (light chain) using Seamless Cloning and Assembly Enzyme Mix (Thermo Fisher Scientific, A14606).

[0144] The resulting vector was used to transform Escherichia coli to obtain transformants, which were then cultured in large quantities. The heavy and light chain plasmids from each clone were purified. Antibodies were transiently expressed using the resulting plasmids and the Expi293 Expression System (Thermo Fisher Scientific, A14635). Antibodies were then purified from each culture supernatant using MabSelect SuRe (Cytiva, 17543802). For stabilization purposes, the core hinge sequence of the human IgG4 heavy chain constant region was substituted from Ser to Pro (Angal S. et al., Molecular Immunology, 1993). The human light chain constant region was a typical κ chain sequence. Using the above-described method, a humanized 3E1C antibody was obtained. The humanized 3E1C antibody contained a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 199 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 10, as shown in Table 7 below.

[0145]

[0146] (2-6) Construction of Humanized 3E1C Mutants Mutants of the humanized 3E1C parent antibody were prepared. Specifically, Cys present in CDR3 of the heavy chain variable region of the humanized 3E1C antibody (3E1C parent antibody) obtained in Example 1 (2-5) was substituted with alanine (A), phenylalanine (F), or valine (V). The substitutions were performed by designing the primers shown in Table 8 below and using a KOD-Plus-Mutagenesis Kit (TOYOBO, SMK-101). After the mutations were introduced, the substituted amino acids were identified by sequence analysis. Table 9 below shows the amino acid sequences of the heavy chain variable regions of the 3E1C parent antibody mutants.

[0147]

[0148]

[0149] The heavy chain expression vectors into which each mutation had been introduced were transiently expressed together with the light chain expression vector for the 3E1C parent antibody obtained in Example 1 (2-5) using the Expi293 Expression System (Thermo Fisher Scientific, A14635) in accordance with the 40 ml protocol. Table 7 shows the amino acid sequence of the light chain variable region of the 3E1C. After the expression, each culture supernatant was purified using MabSelect SuRe (Cytiva, 17543802), and purified antibodies of the humanized 3E1C mutants were obtained.

[0150] (2-7) Study on Degradation of Humanized 3E1C Mutants Next, SDS-PAGE was performed on each of the purified antibodies. Specifically, the purified 3E1C parent antibody obtained in Example 1 (2-5) and the humanized 3E1C mutant obtained in Example 1 (2-6) were adjusted to a final application amount of 1 μg and heated at 70°C for 10 minutes. After the heat treatment, each purified antibody was applied to NuPAGE™ 4 to 12% Bis-Tris, 1.0-1.5 mm (Thermo Fisher Scientific, #NP0329BOX) and electrophoresed at a constant voltage of 110 V for 90 minutes. The electrophoresis buffer used was NuPAGE™ MES SDS Running Buffer (Thermo Fisher Scientific, #NP0002). The gel was stained using Quick-CBB PLUS (Fujifilm Wako Pure Chemical Industries, Ltd., #178-00551). The results are shown in Figure 3.

[0151] Figure 3 shows the results of SDS-PAGE. In Figure 3, the vertical axis represents molecular weight, and the horizontal axis represents the substituted amino acid for each purified antibody. As shown in Figure 3, bands were observed around 150 kDa and between 100 and 150 kDa for the 3E1 parent antibody, whereas bands were observed only around 150 kDa for each purified mutant antibody. These results demonstrate that antibody degradation is suppressed by substituting alanine, phenylalanine, or valine for the cysteine ​​present in CDR3 of the heavy chain variable region of the 3E1 parent antibody.

[0152] (2-8) Study of Affinity of Each Purified Antibody of the Humanized 3E1C Mutants The affinity of each purified antibody of the humanized 3E1C mutants was studied. Specifically, the affinity was measured using single-cycle kinetics. The h3E1C parent antibody obtained in Example 1 (2-5) and each purified antibody of the humanized 3E1C mutant obtained in Example 1 (2-6) were adjusted to 2 nmol / L using running buffer HBS-EP+ buffer (Cytiva, BR100669) to obtain a ligand solution. The antigen huCADM1-NTF-HisTag was diluted with running buffer to prepare five 3-fold dilutions starting from 30 nmol / L to obtain an analyte solution. The running buffer was also used as a blank solution. Next, the ligand solution was added to Flow Cell 2 at a flow rate of 10 μl / min for 60 seconds. After the addition, the blank solution or the analyte solution was added to Flow Cells 1 and 2 at a flow rate of 30 μl / min for 120 seconds. The dissociation time was 900 seconds. Analysis was performed using Biacore™ Insight Evaluation Software (Cytiva), and binding parameters were calculated using a 1:1 binding model. The results are shown in Table 10 below. As shown in Table 10 below, each purified antibody of the humanized 3E1C mutants was found to exhibit high affinity, similar to the 3E1C parent antibody.

[0153]

[0154] (2-9) Examination of the reactivity of each purified antibody of the humanized 3E1C mutant by ELISA The reactivity of each purified antibody of the humanized 3E1C mutant was examined by ELISA. Specifically, 5 μg / ml BSA (T9310A, manufactured by Takara Bio Inc.) was immobilized on an immunoplate (442404, manufactured by Thermo Fisher Scientific) at 4°C overnight. After immobilization, the immobilized solution was removed and the plate was washed with PBS-T (405TS, manufactured by BioTek). After washing, 20% blocking solution (Blocking One, manufactured by Nacalai Tesque Inc., 03953-95) was added and incubated at 37°C for 1 hour. After incubation, the plate was washed with PBS-T. After washing, the primary reaction was carried out at 37°C for 1 hour using the purified 3E1 parent antibody obtained in Example 1 (2-5) and the humanized 3E1 mutant antibody obtained in Example 1 (2-6). The antibody concentration was 100 μg / ml with eight 5-fold dilutions. After the primary reaction, the cells were washed three times with PBS-T, and then a secondary reaction was carried out at 37°C for 1 hour with a secondary antibody (HRP-anti-mouse IgG, Seracare, 5450-0011) diluted to 0.5 μg / ml. After the secondary reaction, the cells were washed three times with PBS-T. After washing, TMB (KPL, 5120-0076) was added, and color development was carried out at room temperature for 20 minutes. After color development, the reaction was stopped by adding TMB stop solution (KPL, 5150-0020). Thereafter, absorbance (450 nm / 650 nm) was detected using a plate reader (Cytation, BioTek). The results are shown in FIG.

[0155] Figure 4 is a graph showing the results of ELISA. In Figure 4, the vertical axis represents absorbance, and the horizontal axis represents the concentration (μg / ml) of the primary antibody. As shown in Figure 4, each purified antibody of the 3E1C mutant was found to have reduced nonspecificity compared to the 3E1C parent antibody.

[0156] (2-10) Determination of Epitope The epitope of the 3E1 antibody on CADM1 was investigated. First, using a peptide fragment of human CADM1 (SEQ ID NO: 1), it was confirmed that the 3E1 antibody binds to the C-terminal region of the third Ig loop of human CADM1 (amino acids 306 to 317 in the amino acid sequence of SEQ ID NO: 1) and the N-terminal region of the juxtamembrane domain (amino acids 318 to 326 in the amino acid sequence of SEQ ID NO: 1). Next, to confirm which amino acid residues in these regions the 3E1 antibody recognizes, mutants (alanine mutants or phenylalanine mutants) were prepared and the epitope was determined. Specifically, mutant CADM1s were prepared having the following mutations in the amino acid sequence of human CADM1 (SEQ ID NO: 1): T306A, T310A, Y311A, R312A, E314A, S316A, K321A, S324A, D325A, Y326A, or Y326F. Western blots were then performed using each mutant CADM1 and the 3E1 antibody, and the binding of each mutant CADM1 to the 3E1 antibody was detected using a film. The signal level on the resulting film was quantified, and the resulting value was used as the binding activity of the 3E1 antibody. Measurements were similarly performed except that wild-type human CADM1 (SEQ ID NO: 1) was used as a control. The binding activity (S) of the 3E1 antibody to mutant CADM1 was then visually evaluated using the binding activity of the 3E1 antibody to wild-type human CADM1 as the standard, according to the following criteria: Furthermore, amino acid residues with binding activity of +++ or ++ were evaluated as having binding to the 3E1 antibody. The results are shown in Table 11 below. (3E1 binding activity) +++: Equivalent to wild-type CADM1 ++: Slightly weaker than wild-type CADM1 +: Significantly weaker than wild-type CADM1 + / -: Slightly reactive -: No reactive

[0157]

[0158] As shown in Table 11, the 3E1 antibody was found to recognize and bind to the amino acids at positions 311, 325, and 326 of human CADM1 (SEQ ID NO: 1). Since the amino acid residue at position 311 of human CADM1 (SEQ ID NO: 1) is modified with a sulfate group (sulfo group), the decrease in binding activity due to the alanine substitution at position 312 is presumed to be due to the absence of sulfate group modification of the amino acid residue at position 311. From the above, it was found that the 3E1 antibody recognizes and binds to the amino acid residues at positions 311, 325, and 326 of human CADM1.

[0159] Example 2 (1) Construction of 9D2 Antibody First, a chicken anti-CADM1 antibody (chicken 9D2 antibody) was produced. The antigen used was a recombinant protein of the full-length extracellular domain of mouse CADM1 (Furuno T. et al., Journal of Immunology, 2005). After immunizing chickens with the antigen, antibody-producing cells were collected from the spleen and fused with the chicken B cell line MuH1. Hybridoma clones producing the anti-CADM1 antibody were selected by ELISA based on their reactivity to CADM1-Fc (Koma Y. et al., Oncogene, 2004), which is a clone in which the Fc domain of mouse IgG has been added to the C-terminus of the extracellular domain of mouse CADM1.

[0160] The nucleic acid sequence encoding the obtained chicken anti-CADM1 antibody was decoded from the hybridoma producing the antibody, and the amino acid sequence was identified from the nucleic acid sequence. As a result, the chicken anti-CADM1 antibody (9D2 antibody) was found to comprise a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 184 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 188. The heavy chain CDRs 1 to 3 and the light chain CDRs 1 to 3 in the chicken anti-CADM1 antibody (9D2 antibody) were identified according to the Kabat numbering system. In the following SEQ ID NOs: 184 and 188, the underlined amino acid sequences represent CDRs 1 to 3 from the N-terminus. 9D2 antibody Heavy chain variable region (SEQ ID NO: 184): AVTLDESGGGLQTPGGALSLVCKASGFTFSDYGMGWMRQAPGKGLEFVAGIDDGGSYIAYGSAVKGRATISRDNGQSTVRLQLNNLRAEDTGTYYCAKASDSGSYSSAAYAGSIDAWGHGTEVIVSS Light chain variable region (SEQ ID NO: 188): ALTQPSSVSANPGETVEITCSGGGRSSYYGWHQQKSPGSAPVTLIYDNTNRPSDIPSRFSGSKSGSTATLTITGVQADDEAVYFCGNEDSSGTAIFGAGTTLTVL

[0161] (2) Humanized 9D2 Antibody and Mutants Thereof Humanized 9D2 antibody and mutants thereof were prepared, and the affinity of the antibodies was examined.

[0162] (2-1) Humanization of the 9D2 Antibody The chicken 9D2 antibody was humanized. Specifically, antibody germlines structurally compatible with the chicken 9D2 variable region were selected in silico, and humanized antibodies were designed using CDR grafting. The design was performed for three heavy chain variable regions and six light chain variable regions. Total synthesis of each designed sequence was outsourced to Eurofins Genomics. Table 12 below shows the amino acid sequences of the humanized variable regions of the 9D2 antibody. The heavy chain variable region and the light chain variable region were each recombined into an expression vector incorporating a human antibody constant region. The resulting plasmids were then transiently expressed using the Expi293 Expression System (Thermo Fisher Scientific, A14635) according to the 25 ml protocol. Each culture supernatant was purified using MabSelect SuRe (Cytiva, 17543802).

[0163]

[0164] (2-2) Study of the Affinity of Humanized 9D2 Antibody The affinity of the humanized 9D2 antibody was studied. Specifically, the affinity was measured using single-cycle kinetics. The antigen biotin-CADM1-Fc was prepared at 10 nmol / L using running buffer HBS-EP+ buffer (Cytiva, BR100669) to obtain a ligand solution. Biotin CAPture Reagent was diluted 10-fold using running buffer. The humanized 9D2 antibody obtained in Example 2 (2-1) above was diluted with running buffer to prepare five 3-fold dilutions starting from 3 nmol / L to obtain an analyte solution. The running buffer was also used as a blank solution. Next, Biotin CAPture Reagent was added to Flow Cell 2 at a flow rate of 2 μl / min for 300 seconds. After this addition, the ligand solution was added at a flow rate of 10 μl / min for 60 seconds. After the addition, the blank solution or the analyte solution was added to Flow Cells 1 and 2 at a flow rate of 30 μl / min for 120 seconds. The dissociation time was 600 seconds. Analysis was performed using Biacore™ Insight Evaluation Software (Cytiva), and binding parameters were calculated using a 1:1 binding model. The results are shown in Table 13 below. As shown in Table 13 below, the humanized 9D2 antibody shown in Table 13 was found to exhibit high affinity.

[0165]

[0166] Example 3 An anti-CADM1 antibody that binds to the same region as the 3E1 antibody was obtained.

[0167] (1) Antibody Acquisition Three 29-day-old chickens (Boris Brown) were used. The antigen concentration was adjusted to 0.3 mg / 1.5 ml / 3 chickens with PBS(-). The antigen was the extracellular domain of mouse CADM1 (SEQ ID NO: 2). After the preparation, the antigen and complete Freund's adjuvant (FCA, Fujifilm Wako Pure Chemical Industries, Ltd.) were mixed at a 1:1 ratio to form an emulsion, which was then administered intraperitoneally to the chickens for primary immunization. Subsequently, the antigen was adjusted to 0.3 mg / 1.5 ml / 3 chickens with PBS(-) to form a 1:1 emulsion with incomplete Freund's adjuvant (FIA, Fujifilm Wako Pure Chemical Industries, Ltd.) to form an emulsion, which was then administered intraperitoneally to the chickens for secondary immunization. Two days before spleen collection, the chickens were administered a final immunization via the underwing vein with antigen prepared in PBS(-) at 0.1 mg / 1 ml per chicken. The spleens were then collected from the chickens, and mRNA was extracted using an mRNA extraction kit (Promega, Z5310). After extraction, cDNA synthesis (Takara Bio, 6210A) was performed from the mRNA, and the variable region genes of the chicken antibody heavy and light chains were amplified by PCR (Toyobo, KOD-401). The resulting gene fragments were then annealed with linkers and further amplified from both ends by PCR. Following amplification, the resulting gene fragments were ligated into a pPDS vector with a 15-bp complementary strand using Gibson assembly. After the Gibson assembly, the buffer was replaced with ultrapure water, and gene transfer was performed into XL1-Blue (Stratagene, 200228) using electroporation (ELEPO21, manufactured by Nepa Gene Co., Ltd.). After the gene transfer, the cells were cultured at 37°C for 5 hours using SOC medium. After the culture, helper phage was added and cultured at 37°C for 1 hour. After the culture, the cells were centrifuged and collected. After the collection, the cells were suspended in 10 ml of 2xYT (final concentrations: 50 μg / ml ampicillin, 25 μg / ml kanamycin, 100 μg / ml IPTG). After the suspension, phage scFv antibody expression was performed overnight at 30°C. The culture supernatant was then collected and filtered.After the filtration, 20% PEG / 2.5 mol / L NaCl was added in an amount of 1 / 5 of the supernatant, stirred, and allowed to stand on ice for 1 hour. After standing, the mixture was centrifuged at 10,000 x g for 30 minutes. After centrifugation, the supernatant was thoroughly removed, and the precipitate was dissolved using PBS to obtain a sample. The sample was stored at 4°C. The sample was used as a primary scFv antibody library.

[0168] The scFv antibody library was subjected to panning using plates immobilized with recombinant CADM1. The panning was performed according to the method described in Reference 1 below. After five rounds of panning, ELISA was performed using plates immobilized with recombinant CADM1 to examine the reactivity of the library. Phage screening was performed from libraries that showed increasing reactivity in the ELISA. For the screening, phages were infected into Escherichia coli and plated on 2xYT agar plates containing 50 μg / ml ampicillin to form colonies. After colony formation, the colonies were cultured in ampicillin-containing 2xYT liquid medium. The colonies were then infected with helper phage, and phage induction was performed using 2xYT liquid medium containing 50 μg / ml ampicillin, 25 μg / ml kanamycin, and 100 μg / ml IPTG. After the induction, the reactivity of the scFv phage antibodies in the resulting culture supernatant was examined by ELISA using an antigen-coated plate. The sequences of the resulting positive clones were determined by sequencing. Furthermore, clones with different sequences were recombined into mouse chimeric forms using the DNA strand encoding the scFv antibody as a template. Based on the resulting amino acid sequences, the heavy chain CDRs 1-3 and light chain CDRs 1-3 of each antibody were identified according to the Kabat numbering system (the same applies below). Tables 2A-C show the heavy chain variable regions and light chain variable regions of the resulting clones, as well as the amino acid sequences of the heavy chain CDRs 1-3 and light chain CDRs 1-3. Reference 1: Nakamura N, Shuyama A, Hojyo S, Shimokawa M, Miyamoto K, Kawashima T, Aosasa M, Horiuchi H, Furusawa S, Matsuda H. Establishment of a chicken monoclonal antibody panel against mammalian prion protein. J Vet Med Sci. 2004 Jul;66(7):807-14. doi: 10.1292 / jvms.66.807. PMID: 15297752.

[0169] (2) Examination of ELISA Reactivity of Newly Obtained Antibodies The reactivity of newly obtained antibodies was examined by ELISA. Specifically, antigen (mouse CADM1, BSA) prepared at 5 μg / ml was immobilized on an immunoplate (Thermo Fisher Scientific, 442404) overnight at 4°C. After immobilization, the solid phase solution was removed, and 20% blocking solution (Blocking One, Nacalai Tesque, 03953-95) was added and incubated at 37°C for 1 hour. After incubation, the plate was washed with PBS-T. After washing, the purified antibodies obtained in Example 3(1) were used as primary antibodies and subjected to a primary reaction at 37°C for 1 hour. The antibody concentrations were 1000 ng / ml and eight 5-fold dilutions were used. After the primary reaction, the plate was washed three times with PBS-T and then incubated at 37°C for 1 hour with a secondary antibody (HRP-anti-mouse IgG, Seracare, 5450-0011) diluted to 0.2 ng / ml. After the secondary reaction, the plate was washed three times with PBS-T. After washing, TMB (KPL, 5120-0076) was added, and color development was carried out at room temperature for 30 minutes. After color development, TMB stop solution (KPL, 5150-0020) was added to terminate the reaction. Absorbance (450 nm / 650 nm) was then detected using a plate reader (Cytation, BioTek). The results are shown in Figures 5 and 6.

[0170] 5 and 6 are graphs showing the results of ELISA. In these figures, the vertical axis represents absorbance, and the horizontal axis represents the concentration (ng / ml) of the primary antibody. As shown in FIGS. 5 and 6, all of the anti-CADM1 antibodies exhibited concentration-dependent binding to mouse CADM1.

[0171] (3) Humanization of Newly Obtained Antibodies 45TG23 obtained in Example 3(2) above was humanized. Specifically, four humanized sequences were designed for each of the heavy and light chains using in silico analysis. The amino acid sequences of the humanized sequences are shown in Table 14 below. Based on the designed amino acid sequences, polynucleotides encoding the heavy and light chains were totally synthesized. The heavy chain was then recombined into an expression vector incorporating the human IgG4 constant region, and the light chain was then recombined into an expression vector incorporating the human κ constant region. The prepared heavy and light chain plasmids were then combined and transiently expressed using the Expi293 Expression System (Thermo Fisher Scientific, A14635) according to the 30 ml protocol. Each culture supernatant was purified using MabSelect SuRe (Cytiva, 17543802). The amino acid sequences of the combined antibodies are shown in Tables 2A to 2C. In the clone name (A-B) of each antibody, A is the clone name of the heavy chain in Table 14 below, and B is the clone name of the light chain in Table 14 below.

[0172]

[0173] (5) Study of the Affinity of Humanized 45TG23 Antibody The affinity of the humanized 45TG23 antibody was studied. Specifically, the affinity was measured using single-cycle kinetics. The test antibody was adjusted to 2 nmol / L using running buffer HBS-EP+ buffer (Cytiva, BR100669) to obtain a ligand solution. huCADM1-NTF-HisTag was diluted with running buffer to prepare five 3-fold dilutions starting from 30 nmol / L to obtain an analyte solution. The running buffer was also used as a blank solution. Next, the ligand solution was added to Flow Cell 2 at a flow rate of 10 μl / min for 60 seconds. After the addition, the blank solution or the analyte solution was added to Flow Cells 1 and 2 at a flow rate of 30 μl / min for 120 seconds. The dissociation time was 900 seconds. The analysis was performed using Biacore™ Insight Evaluation Software (Cytiva), and binding parameters were calculated using a 1:1 binding model. The results are shown in Table 15 below. As shown in Table 15 below, the humanized 45TG23 antibody was found to exhibit high affinity.

[0174]

[0175] (3) Study of the epitope of anti-CADM1 antibodies The binding site on CADM1 of the antibodies obtained in Example 3(2) above was confirmed. Specifically, mutant CADM1s were prepared containing the Delta3 mutant CADM1, DeltaL3 mutant CADM1, and DeltaY mutant CADM1 mutations, in which a portion of the extracellular domain of mouse CADM1 (SEQ ID NO: 2) was deleted. The binding activity of each antibody was evaluated in the same manner as in Example 1(2-10) above, except that the 43XL17 antibody, 45TG23 antibody, and 43TG47 antibody were used in combination with each mutant CADM1. As a result, the binding activity of each antibody was reduced when any of the mutant CADM1s was used, indicating that the newly obtained anti-CADM1 antibodies bind to the same region as the 3E1 antibody. Delta3 mutated CADM1 (SEQ ID NO: 204): MASAVLPSGSQCAAAAAVAAAAAPPGLRLRLLLLLLSAAALIPTGDGQNLFTKDVTVIEGEVATISCQVNKSDDSVIQLLNPNRQTIYFRDFRPLKDSRFQLLNFSSSELKVSLTNVSISDEGRYFCQLYTDPPQESYTTITVLVPPRNLMIDIQKDTAVEGEEIEVNCTAMASKPATTIRWFKGNKELKGKSEVEEWSDMYTVTSQLMLKVHKEDDGVPVICQVEHPAVTGNLQTQRYLEVQYKAHSDYMLYVYDSRAGEEGTIGAVDH DeltaL3 mutated CADM1 (SEQ ID NO: 205): MASAVLPSGSQCAAAAAVAAAAAPPGLRLRLLLLLLSAAALIPTGDGQNLFTKDVTVIEGEVATISCQVNKSDDSVIQLLNPNRQTIYFRDFRPLKDSRFQLLNFSSSELKVSLTNVSISDEGRYFCQLYTDPPQESYTTITVLVPPRNLMIDIQKDTAVEGEEIEVNCTAMASKPATTIRWFKGNKELKGKSEVEEWSDMYTVTSQLMLKVHKEDDGVPVICQVEHPAVTGNLQTQRYLEVQYKPQVHIQMTYPLQGLTREGDAFELTCCEASNIVGKAHSDYMLYVYDSRAGEEGTIGAVDH DeltaY mutation CADM1 (SEQ ID NO: 206):MASAVLPSGSQCAAAAAVAAAAAPPGLRLRLLLLLLSAAALIPTGDGQNLFTKDVTVIEGEVATISCQVNKSDDSVIQLLNPNRQTIYFRDFRPLKDSRFQLLNFSSSELKVSLTNVSISDEGRYFCQLYTDPPQESYTTITVLVPPRNLMIDIQKDTAVEGEEIEVNCTAMAS KPATTIRWFKGNKELKGKSEVEEWSDMYTVTSQLMLKVHKEDDGVPVICQVEHPAVTGNLQTQRYLEVQYKPQVHIQMTYPLQGLTREGDAFELTCEAIGKPQPVMVTWVRVDDEMPQHAVLSGPNLFINNLNKTDNGTYRCEASNIVGKAHSDMLYVYDSRAGEEGTIGAVDH

[0176] Next, the binding activity of the 45TG23 antibody was evaluated as in Example 1(2-10) using the mutant CADM1 (Y311A, R312A, E314A, S316A, K321A, S324A, D325A, or Y326A) prepared in Example 1(2-10). As a result, it was found that, in the case of the 45TG23 antibody, the 3E1 antibody recognizes and binds to amino acid residues at positions 311, 325, and 326 of human CADM1. Furthermore, as with the 3E1 antibody, the binding activity of the 45TG23 antibody was reduced due to the alanine substitution at position 312, which was presumed to be due to the same reason as for the 3E1 antibody.

[0177] From the above, it was found that the newly obtained antibody obtained in Example 3 recognizes the same region as the 3E1 antibody. In addition, it was found that the 45TG23 antibody recognizes the same epitope as the 3E1 antibody.

[0178] [Example 4] The function of anti-CADM1 antibodies was examined.

[0179] (1) Examination of the Internalization-Promoting Effect of Various Humanized Clones of Anti-CADM1 Antibodies The present inventors found that, in a standard culture system of CADM1-expressing cells, addition of an anti-CADM1 antibody to the culture medium resulted in CADM1 disappearing from the readily soluble fraction and transferring to the poorly soluble protein fraction (Figure 7). This is thought to indicate that CADM1 disappeared from the cell membrane and was transferred into the cells (internalized). Therefore, the internalization efficiency of various humanized clones of anti-CADM1 antibodies that showed high affinity for CADM1 recombinant protein (antigen) in ELISA was examined.

[0180] (1-1) Preparation of various humanized clones of anti-CADM1 antibodies Humanized 3E1#2 antibody (antibody A#2) and 3E1#3 antibody (antibody A#3) were prepared in the same manner as in the preparation of humanized 3E1C antibody (antibody A#1), a humanized clone of anti-CADM1 antibody, in Example 1 above. These antibodies comprised a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 207 of the humanized 3E1#2 antibody and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 208. These antibodies comprised a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 209 of the 3E1#3 antibody and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 210. #2 heavy chain variable region (SEQ ID NO: 207): EVQLLESGGGLVQPGGSLRLSCAASGFTFS[SNAMG]WVRQAPGKGLEFVA[GIGNTGRYTGYGPAVKG]RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK[SASGSGYGCAGWIDA]WGQGTLVTVSS #2 light chain variable region (SEQ ID NO: 208): SYELTQPPSVSVSPGQTARITC[SGGSYIYG]WYQQKPGQAPVTVIY[SNDKRPS]GIPERFSGSTSGSTTTLTISGVQAEDEADYYC[GNEDNSIDSAI]FGGGTKLTVL #3 heavy chain variable region (SEQ ID NO: 209): EVQLLESGGGLVQPGGSLRLSCAASGFTFS[SNAMG]WVRQAPGKGLEWVA[GIGNTGRYTGYGPAVKG]RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK[SASGSGYGCAGWIDA]WGQGTLVTVSS #3 light chain variable region (SEQ ID NO: 210): SYELTQPPSVSVSPGQTARITC[SGGSYIYG]WYQQKPGQAPVTVIY[SNDKRPS]GIPERFSGSNSGSTGTLTISGVQAEDEADYFC[GNEDNSIDSAI]FGGGTKLTVL

[0181] (1-2) Examination of the Internalization-Promoting Effect of Various Clones of Humanized 3E1 Antibody Specifically, the human leukemia cell line MT-2 was cultured, and various clones of the humanized 3E1 antibody (the 3E1 parent antibody (antibody A) obtained in Example 1 (2-6) above, 3E1#1 and 3E1#2 obtained in Example 4 (1-1) above, each at 2 μg / ml) or a control antibody (human IgG (Normal human IgG whole molecule, Wako, Cat. No.: 143-09501): 2 μg / ml)) were added to the culture medium along with the 9D2 antibody (antibody B, chicken IgM: 2 μg / ml) obtained in Example 2 (1) above. The day after the addition, the easily soluble and poorly soluble protein fractions were subjected to protein extraction and Western blotting. The results are shown in Figure 8.

[0182] In Figure 8, the left vertical axis indicates the type of antibody used for detection, the right vertical axis indicates molecular weight (kDa), and the horizontal axis indicates the type of sample. In Figure 8, the blot of human IgG (heavy chain) shows the degree of internalization of the 3E1 antibody clone (human IgG). GAPDH is a marker for the easily soluble fraction, and HDAC is a marker for the poorly soluble protein fraction (including nuclear proteins). As shown in Figure 8, the internalization efficiencies of the 3E1#1 antibody and the 3E1#2 antibody were similarly high. Furthermore, the internalization efficiencies of the 3E1#1 antibody and the 3E1#2 antibody were higher than that of the 3E1#3 antibody.

[0183] (2) Study of healing in an atopic dermatitis model using anti-CADM1 antibodies Figure 9 shows nerve-mast cell interactions mediated by CADM1. Figure 9(a) shows mast cells adhering to peripheral nerve fibers. In peripheral interstitial tissues such as the dermis, mast cells sometimes exist in contact with peripheral nerve fibers. For example, CADM1 immunostaining reveals that both nerves and mast cells express CADM1. In Figure 9(a), the hatched areas indicate areas that were positive for CADM1 immunostaining. Because CADM1 is an adhesion molecule that binds in trans to homologous cells, it is thought to directly mediate nerve-mast cell adhesion.

[0184] In atopic dermatitis models, CADM1 expression in mast cells is enhanced, which is thought to result in enhanced neuronal-mast cell interactions. Substance P ("SP" in Figure 9(b)), secreted by nerves, is known to be an effector of functional interactions between the two. Enhanced interactions are thought to significantly contribute to the formation of these lesions by inducing itching and prolonging inflammation. Therefore, the present inventors hypothesized that anti-CADM1 antibodies might contribute to the healing of atopic dermatitis models by reducing and normalizing excessive neuronal-mast cell interactions within skin lesions. Therefore, they investigated whether anti-CADM1 antibodies could be used to heal atopic dermatitis models.

[0185] Specifically, the drug Biosta (a reagent containing a house dust mite-derived allergen) was applied to NC / Nga mice (16 weeks old, female) to create an atopic dermatitis model. The therapeutic effect of anti-CADM1 antibodies was then examined using this atopic dermatitis model. The atopic dermatitis model was created and the antibody was administered as follows. Figure 10 shows an overview of the creation of the atopic dermatitis model and antibody administration.

[0186] <Procedure> 1) The dorsal skin of NC / Nga mice was exposed using a hair removal cream. 2) Biosta was applied to the exposed area twice a week for three consecutive weeks, a total of six times. 3) The NC / Nga mice were divided into groups so that the severity of dermatitis was consistent. 4) On the day of grouping, the following day, and the day after that, the 3E1 antibody (chicken-mouse chimeric antibody) obtained in Example 1 (1-1) or a control antibody (mouse IgG1, Mouse IgG1 isotype control, AcroBiosystems, Cat. No.: DNP-M1) was injected subcutaneously into the skin lesions for three consecutive days. 10 μg of antibody was administered per mouse.

[0187] Regarding the severity of dermatitis, skin lesions were observed on the day of grouping and 12 days later, and various symptoms (dryness, crusting, bleeding, abrasions, etc., four categories A to D; Figure 10) were scored (0, no findings; 1, mild; 2, moderate; 3, severe), and the sum of these scores was used as the dermatitis score. (Dermatitis score on the day of grouping) - (score 12 days later) was used as the degree of dermatitis improvement. These results are shown in Figure 11 and Table 3 below.

[0188] Figure 11 shows photographs of dermatitis in an atopic dermatitis model. Table 16 below shows the results of calculations of the mean ± standard deviation of the degree of improvement in dermatitis score for each group, using six mice per group. P values ​​in comparison with the control antibody group were calculated using Student's t-test. As shown in Table 16 below, the degree of improvement in dermatitis score was found to be significantly increased in the 3E1 antibody-administered group.

[0189]

[0190] Furthermore, because this dermatitis model is accompanied by a strong itching sensation, mice frequently scratch the lesions. We investigated whether administration of an anti-CADM1 antibody inhibits this scratching behavior. The behavior of the mice was videotaped for 30 minutes on the day of grouping, and 4 and 5 days after grouping. The video was observed, and the total time spent scratching the lesions was calculated. The scratching behavior time on the day of grouping was set to 1 for each individual mouse, and a relative value was calculated. The mean ± standard deviation of the scratching behavior time 4 and 5 days later is shown in Table 17 below. P values ​​in comparison with the control antibody group were calculated using the Mann-Whitney U test. As shown in Table 17 below, the scratching behavior time was significantly reduced in the 3E1 antibody-administered group.

[0191]

[0192] (3) Study of Intracranial Drug Delivery Using Anti-CADM1 Antibodies The inner surface of the nasal cavity in mice and humans is covered by respiratory epithelium or olfactory epithelium. The olfactory epithelium occupies the deep region of the nasal cavity, i.e., the region closest to the brain. The olfactory epithelium is composed of olfactory receptor cells and supporting cells, and nerve fibers extending from the olfactory receptor cells penetrate the ethmoid bone (a bone at the base of the skull) and reach the olfactory bulb (part of the rhinencephalon) within the skull. CADM1 is known to be highly expressed in the olfactory receptor cells and supporting cells. Figure 12 shows the expression of CADM1 in the nasal mucosa. In Figure 12, (a) shows the structure of the mouse forehead (sagittal section), and (b) to (d) show enlarged views of the region from the deep nasal cavity to the rhinencephalon. Specifically, Figure 12 shows the results of immunostaining the forehead of an adult C57BL / 6 mouse using an antibody recognizing the C-terminus of CADM1. In Figure 12, hatched areas indicate CADM1-positive areas. As shown in Figure 12, CADM1 is highly expressed in the olfactory epithelium. On the other hand, CADM1 is hardly expressed in the respiratory epithelium. Therefore, we investigated whether anti-CADM1 antibodies could be delivered intracranially (near the olfactory bulb).

[0193] Specifically, anti-CADM1 antibodies were administered to adult C57BL / 6 mice via nasal instillation once daily for two consecutive days. The anti-CADM1 antibodies used were the chicken 3E1 antibody obtained in Example 1 (1-1) and the chicken 9D2 antibody obtained in Example 2 (1). A control antibody (Normal chicken IgY control, R&D, Cat. No.: AB-101-C) was used as a control. The nasal instillation doses were 10 μg each for the chicken 3E1 antibody and the chicken 9D2 antibody. On the third day, the frontal regions of the mice were removed and immunostained using anti-chicken IgY antibody. The results are shown in Figure 13.

[0194] Figure 13 shows the results of immunostaining of the mouse forehead. In Figure 13, the hatched area indicates the detection area of ​​the anti-CADM1 antibody. As shown in Figure 13, the nasally instilled anti-CADM1 accumulated in the olfactory epithelium and was localized across the ethmoid bone into the skull. Anti-CADM1 antibody was also localized within the nerve fibers extending from olfactory cells and within the rhinencephalon, where these nerve fibers terminate. Although not shown, the control antibody diffused and disappeared within approximately 30 minutes, whereas the anti-CADM1 antibody accumulated in the olfactory epithelium and remained at high concentrations even after 3 hours. These results demonstrate that when anti-CADM1 antibody is instilled into the nasal cavity of a mouse, the anti-CADM1 antibody accumulates in the olfactory epithelium and remains there for a long time, and some of the anti-CADM1 antibody reaches the skull (near the olfactory bulb). It was suggested that anti-CADM1 antibodies can provide a drug delivery vector for intracranial delivery of target drugs via CADM1 expressed in the olfactory epithelium.

[0195] (4) Intracranial Delivery of Nasally Instilled Anti-CADM1 Antibody The intracranial delivery of nasally instilled anti-CADM1 antibody was investigated. Specifically, anti-CADM1 antibody was administered to mice. In the administration, mice No. 1 and No. 2 were administered 10 μg of the chicken 3E1 antibody obtained in Example 1 (1-1) and the chicken 9D2 antibody obtained in Example 2 (1), respectively. In the administration, mice No. 3 were administered 10 μg of the chicken 3E1 antibody obtained in Example 1 (1-1) alone. A control antibody (chicken IgY, Normal chicken IgY control, R&D, Cat. No.: AB-101-C) was used as a control. Mice that did not receive nasal instillation served as a negative control. After the nasal instillation, the mouse forehead was removed, embedded in Tissue-Tek OCT compound (Sakura Finetek, Torrance), frozen, and cryosectioned using a cryostat. Figure 14 shows microscopic images of the nasal cavity and rhinencephalon regions in the cryosections from the control and anti-CADM1 antibody-treated groups. Proteins were extracted from the nasal cavity and rhinencephalon regions, respectively. After the extraction, Western blotting was performed on the extracted proteins. Antibodies (heavy chains) and various markers (epithelial markers, neural markers, and cell mass markers) were used for the Western blotting. The results are shown in Figure 15.

[0196] Figure 15 is a photograph showing the results of Western blotting of proteins in the nasal cavity and rhinencephalon in mice administered nasal instillation of anti-CADM1 antibody. In Figure 15, the left vertical axis indicates the type of antibody used for detection, the right vertical axis indicates molecular weight (kDa), and the horizontal axis indicates the type of sample. Epithelium is present in the nasal cavity region but not in the rhinencephalon region. Nerves are present in both the nasal cavity region and the rhinencephalon region, but are more abundant in the rhinencephalon region. As shown in Figure 14, the blots for epithelial markers and neural markers demonstrate accurate differentiation between the nasal cavity region and the rhinencephalon region. As shown in Figure 15, almost equal amounts of the control antibody and chicken 3E1 antibody were detected in the nasal cavity region. Only chicken 3E1 antibody was detected in the rhinencephalon region. The results for No. 3 mouse suggest that sufficient internalization of the chicken 3E1 antibody occurs when administered nasally, even in the absence of chicken 9D2 antibody.

[0197] Figure 16 is a schematic diagram showing nose-to-brain delivery (see Mariko Takeda, "Current Status and Future of Nasal Drug Delivery" (Japanese Journal of Pharmacology)). As shown in Figure 16, when attempting to deliver a drug intracranially via the nose, the following two delivery routes are considered, taking advantage of the anatomical and histological characteristics of the olfactory epithelium: 1) Taken up by olfactory cells → Transported into the skull by axons → Released extracellularly (solid arrow); 2) Diffusion between olfactory epithelial cells → Penetrates supporting cells → Passes through the axon-ethmoid space → Enters the skull (dashed arrow). In other words, there are two possible routes to the target: 1) After being taken up into olfactory cells, the antibody is transported within olfactory cell nerve fibers toward the olfactory bulb; 2) Diffusion passes through the olfactory epithelium layer and the nerve fiber penetration section of the ethmoid bone.

[0198] From the results of Example 4(4) above, it was found that CADM1 is highly expressed in both the olfactory cells and supporting cells that make up the olfactory epithelium. Therefore, the inventors of the present application discovered that by utilizing CADM1 to accumulate various molecules, such as anti-CADM1 antibodies or complexes of anti-CADM1 antibodies and drugs, in the olfactory epithelium, these molecules can be efficiently delivered intracranially.

[0199] (5) Study of Nasal Intracranial Drug Delivery The nasal intracranial drug delivery of an anti-CADM1 antibody was studied. Specifically, the antibody moiety of the microtubule polymerization inhibitor (MMAE)-conjugated anti-CD30 monoclonal antibody Adcetris (brentuximab vedotin; a therapeutic agent for malignant lymphoma) was replaced with the 3E1 antibody (chicken-mouse chimeric antibody) obtained in Example 1 (1-1) above or a control antibody (mouse IgG1, Mouse IgG1 isotype control, AcroBiosystems, Cat. No.: DNP-M1) to prepare antibody-drug conjugates (hereinafter referred to as "3E1 antibody-MMAE conjugate" or "control antibody-MMAE conjugate"). Next, B16-F10 melanoma cells (cell number: 3 × 10) were intracranially transfected into B57BL / 6 mice. 6 (100 pieces) were transplanted. After the transplantation, a combination of the 3E1 antibody (antibody A)-MMAE conjugate and the 9D2 antibody (antibody B) obtained in Example 2(1) above (hereinafter referred to as "3E1 antibody-MMAE conjugate + 9D2 antibody") was administered nasally from day 4 to day 7 after the transplantation. The nasal administration was performed four times in total, with 10 μg of both the conjugate and the antibody administered per administration. In the control group, an equivalent volume of PBS was administered nasally on the same day. Five hours after the final nasal administration, pathological specimens were prepared and subjected to TUNEL staining. The results are shown in Figure 17 and Table 18 below.

[0200] Figure 17 is a photograph showing the results of TUNEL staining. In Figure 17, (a) shows the results for the control group, and (b) shows the results for the group to which the 3E1 antibody-MMAE conjugate + 9D2 antibody was administered nasally. In Figure 17, black circles indicate TUNEL-positive areas. As shown in Figure 17, there were more TUNEL-positive areas in the group to which the 3E1 antibody-MMAE conjugate + 9D2 antibody was administered nasally than in the control group.

[0201]

[0202] The table 18 is 1 mm ２ The number of TUNEL-positive cells per 1 mm nasal instillation (n=7 for each group) was calculated and the results are shown in Table 18. The P value for the comparison with the control antibody group was calculated by Student's t-test. As shown in Table 18, compared to the control group, the group administered with 3E1 antibody-MMAE conjugate + 9D2 antibody had a significantly higher TUNEL-positive cell count per 1 mm nasal instillation.２ The number of TUNEL-positive cells per nasal cavity was significantly higher. These results indicate that apoptosis was induced in the group treated with the 3E1 antibody-MMAE complex plus the 9D2 antibody. After B16-F10 melanoma cells were implanted intracranially in mice, nasal administration of the 3E1 antibody-MMAE complex plus the 9D2 antibody presumably delivered MMAE intracranially, inducing apoptosis in the melanoma cells.

[0203] (6) Study of brain tumor treatment by anti-CADM1 antibody administration We investigated whether anti-CADM1 antibody administered via nasal instillation is effective in treating brain tumors. Specifically, the luciferase gene was introduced into the glioma cell line CT-2A cells, and cells (CT-2A-Luc) that constitutively express luciferase were obtained. ５CT-2A-Luc cells were intracranially transplanted into 33 female C57BL / 6 mice (6 weeks old) (day 0). Nasal administration of the antibody (phosphate buffer solution) began on day 2 in 17 mice (antibody nasal administration group). Nasal administration was performed every weekday (Monday to Friday), once in the morning and once in the afternoon. The antibody doses administered per administration were 10 μg of the 3E1 antibody (chicken-mouse chimeric antibody) and MMAE conjugate (average DAR 4) obtained in Example 1 (1-1) above, and 10 μg of the 9D2 antibody (chicken-mouse chimeric IgM antibody). The chicken-mouse chimeric IgM antibody was prepared by conjugating the Fab of the 9D2 antibody (chicken antibody) with mouse IgM Fc. Nasal administration was continued until the mice weighed 12 g or less (maximum 14 days). Of the 33 mice, 16 (control group) received nasal instillation of phosphate buffer at the same time. Luciferin was intraperitoneally injected on days 4, 9, and 14 after the nasal instillation, and the luciferin luminescence intensity in the mouse head was measured 15 minutes later using the Pearl Trilogy system. The luciferin luminescence measurement value on day 4 was set to 1, and a relative value was calculated for each mouse. Table 19 below shows the mean ± standard deviation of the luciferin luminescence measurements on days 9 and 14. The number in parentheses indicates the number of mice. Note that a certain number of mice died due to tumor growth by day 14, so the number of mice on day 14 decreased. P values ​​compared with the control antibody group were calculated using Student's t-test. As shown in Table 19 below, the relative measurement value was significantly reduced in the group treated with the antibody nasally compared to the control group. These results suggest that the 3E1 antibody-MMAE conjugate is effective as a nasal treatment for brain tumors.

[0204]

[0205] (7) Study on Delivery of Anti-CADM1 Antibody to Peripheral Neurons, etc. The delivery of anti-CADM1 antibody to peripheral neurons, mast cells, and osteoblasts was studied. Specifically, the 3E1 antibody (chicken-mouse chimeric antibody, chicken IgY: 10 μg) obtained in Example 1 (1-1) above was subcutaneously injected into the back of a mouse. Five hours after the subcutaneous injection, the skin at the subcutaneously injected site was excised. After the excision, immunostaining was performed using an anti-chicken IgY antibody (AffiniPure™ Rabbit Anti-Chicken IgY (IgG), Fc fragment specific, Jackson ImmunoResearch, Cat. No.: 303-005-008). The results are shown in Figure 18.

[0206] Figure 18 is a photograph showing the results of immunostaining. In Figure 18, the area surrounded by a dashed line is the IgY-positive area. As shown in Figure 18, the 3E1 antibody was found to accumulate widely in peripheral nerve fibers (thin stained areas) as well as in mast cells (triangle arrows) and some hair follicle epithelial cells (arrows). The accumulation sites of the 3E1 antibody closely matched the expression distribution of CADM1, indicating that the 3E1 antibody has a very high affinity for CADM1. These results suggest that the 3E1 antibody is useful as a drug delivery vector for CADM1-expressing cells. Furthermore, antibody accumulation on the cell membrane may alter cellular function and exert pharmacological effects. This suggests that locally administered 3E1 antibody may accumulate in CADM1-expressing cells in tissues and exert pharmacological effects.

[0207] (8) Study on Delivery of Anti-CADM1 Antibody to Mast Cells The delivery of anti-CADM1 antibody to mast cells was studied. Specifically, 50 μl of 1% TNCB was applied to the abdomen of NC / Nga mice (sensitization). Four days after application, the 3E1 antibody (chicken-mouse chimeric antibody) obtained in Example 1 (1-1) above or a control antibody (mouse IgG1, Mouse IgG1 isotype control, AcroBiosystems, Cat. No.: DNP-M1) was subcutaneously injected (10 μg each) into the base of the ear. Five hours and the following day after the subcutaneous injection, 20 μl of 1% TNCB was instilled into the ear (challenge). Three hours after the challenge, the ear was removed and immunostained using an anti-tryptase antibody. The results are shown in Figure 19 and Table 20 below.

[0208] Figure 19 is a photograph showing the results of immunostaining. In Figure 19, the arrow indicates a non-degranulated mast cell. In Figure 19, the area surrounded by a dashed line indicates a degranulated mast cell. As shown in Figure 19, the group subcutaneously injected with the 3E1 antibody had a lower number of degranulated mast cells than the control antibody group.

[0209]

[0210] Table 20 shows the results of counting the number of degranulated mast cells per 100 tryptase-positive mast cells in a total of four experiments, and calculating the mean ± standard deviation (%) of the percentage of degranulated mast cells from the calculated values ​​for each of the four experiments. P values ​​for comparison with the control antibody group were calculated using Student's t-test. As shown in Table 20, the number of degranulated mast cells was significantly lower in the group subcutaneously injected with the 3E1 antibody compared to the control group. These results suggest that the 3E1 antibody accumulates on the mast cell membrane and thereby suppresses increased mast cell degranulation in immediate-type allergic reactions. The mechanism is thought to be inhibition of IgE receptor cross-linking on the mast cell membrane.

[0211] (9) Study of Heat Avoidance Behavior by Administration of Anti-CADM1 Antibody B57BL / 6 mice were subcutaneously injected with the 3E1 antibody (chicken-mouse chimeric antibody) obtained in Example 1 (1-1) above, or a control antibody (mouse IgG1, Mouse IgG1 isotype control, AcroBiosystems, Cat. No.: DNP-M1) into the palms and soles of their feet. 10 μg of each antibody was subcutaneously injected into the palms and soles of their feet on both sides. Five hours after the subcutaneous injection, the mice were placed on a hot plate (paraffin spreader Model PS-51, Sakura Seiki) maintained at 52°C, and the time until they exhibited heat avoidance behavior (licking the palm or sole) was measured. The results are shown in Table 21 below.

[0212]

[0213] Table 21 shows the average time (seconds) ± standard deviation for each group. P values ​​for comparison with the control antibody group were calculated using Student's t-test. As shown in Table 21, the time until heat avoidance behavior was significantly longer in the group subcutaneously injected with the 3E1 antibody than in the group subcutaneously injected with the control antibody.

[0214] These results demonstrate that the 3E1 antibody has the potential to be used as a local anesthetic or a systemic peripheral neuralgia reliever. Routes of administration include local administration to the affected area, the epidural space, and intravenous administration. Furthermore, because the 3E1 antibody is highly efficiently internalized, it may be useful as a drug delivery vector for peripheral nerves, potentially serving as a vector for neuroprotective drugs and nucleic acid drugs. Furthermore, by preparing an antibody-drug conjugate loaded with a local anesthetic (e.g., procaine) and administering it subcutaneously or epidurally, it is possible that the anesthetic's effects may be exerted for a very long period of time. In other words, anti-CADM1 antibodies are expected to be long-acting local anesthetics.

[0215] (10) Study on Delivery of Anti-CADM1 Antibody to Osteoblasts The delivery of anti-CADM1 antibody to osteoblasts was studied. Specifically, 5 μg of the 3E1 antibody (chicken-mouse chimeric antibody) obtained in Example 1 (1-1) above was injected subcutaneously near the dorsal vertebral body of newborn B57BL / 6 mice. Five hours after the subcutaneous injection, the mice were fixed in formalin and immunostained using the anti-chicken IgY antibody. These results are shown in Figure 20.

[0216] Figure 20 is a photograph showing the results of immunostaining. In Figure 20, the area surrounded by a dashed line is the IgY-positive area, and the area surrounded by a dashed line is the vertebral bone tissue. The enlarged photograph in Figure 20 shows a magnified view of a portion including the IgY-positive area. As shown in Figure 20, accumulation of the 3E1 antibody was observed in osteoblasts on the vertebral surface and in the trabecular bone within the vertebral body. Furthermore, accumulation of the 3E1 antibody on the cell membrane was observed in many osteoblasts. These results demonstrated that when the 3E1 antibody was locally injected near the bone tissue of mice, the 3E1 antibody accumulated in osteoblasts. This is thought to be due to the high expression of CADM1 in osteoblasts and the high affinity of the 3E1 antibody for CADM1. By utilizing the accumulation property of the 3E1 antibody, the 3E1 antibody was found to be useful as a vector for osteoblast-specific drug delivery. Therefore, it is possible to use the 3E1 antibody to produce antibody-drug conjugates loaded with bone formation-promoting drugs (e.g., estrone) or nucleic acid drugs, which are expected to become therapeutic agents for osteoporosis, intractable fractures, and pseudoarthrosis.

[0217] (11) Investigation of CADM1 Elimination by Anti-CADM1 Antibody As mentioned above, the present inventors found that when anti-CADM1 antibody was added to the culture medium in a standard culture system of CADM1-expressing cells, CADM1 disappeared from the easily soluble fraction and transferred to the poorly soluble protein fraction ( Figure 10 ). This was considered to indicate that CADM1 disappeared from the cell membrane and was transferred into the cell (internalized). Therefore, we investigated whether CADM1 was eliminated by anti-CADM1 antibody using Western blotting. Specifically, various cells (mesothelioma (three types), lung adenocarcinoma, endometrial adenocarcinoma, and small cell lung cancer) were cultured, and the 3E1 antibody (chicken-mouse chimeric antibody) (2 μg / ml) obtained in Example 1(1-1), the chicken 9D2 antibody (2 μg / ml) obtained in Example 2(1), the 3E1 antibody (chicken-mouse chimeric antibody) obtained in Example 1(1-1) and the chicken 9D2 antibody obtained in Example 2(1) (each at 1 μg / ml), or a control antibody (2 μg / ml) was added to the culture medium. The following day, the cells were collected and lysed using a lysis solution (50 mmol / l Tris-HCl (pH 8.0), 150 mmol / l NaCl, 1% Triton X-100). After lysis, the cells were centrifuged, and the supernatant was collected to obtain a readily soluble fraction. Protein extraction (mainly cell membrane proteins and cytoplasmic soluble proteins) was performed on the readily soluble fraction. After the extraction, the samples were subjected to Western blotting, and the results are shown in FIG.

[0218] Figure 21 is a photograph showing the results of Western blotting. In Figure 21, the left vertical axis indicates the type of antibody used for detection, the right vertical axis indicates molecular weight (kDa), and the horizontal axis indicates the type of sample. As shown in Figure 21, for all cells, when both the 3E1 antibody and the 9D2 antibody were added, CADM1 was almost completely eliminated from the easily solubilized fraction. These results demonstrate that CADM1 can be eliminated by the 3E1 antibody and the 9D2 antibody.

[0219] (12) Study of CADM1 Internalization by Anti-CADM1 Antibody Next, we investigated whether CADM1 is internalized by anti-CADM1 antibody. Specifically, the protein pellet generated when obtaining the easily solubilized fraction in Example 4(11) was dissolved in a detergent-containing buffer (RIPA buffer containing 0.1 w / v% sodium dodecyl sulfate) to obtain a poorly soluble protein fraction (containing nuclear proteins). The poorly soluble protein fraction and the easily solubilized fraction obtained in Example 4(11) were subjected to Western blotting. The results are shown in Figure 22.

[0220] Figure 22 is a photograph showing the results of Western blotting. In Figure 22, the left vertical axis indicates the type of antibody used for detection, the right vertical axis indicates molecular weight (kDa), and the horizontal axis indicates the type of sample. In Figure 22, heavy chain indicates the degree of internalization of the blot, GAPDH indicates the easily solubilized fraction, and Lamin B indicates the poorly soluble protein fraction (including nuclear proteins). As shown in Figure 22, when both the 3E1 antibody and the 9D2 antibody were added to both cells, CADM1 was not observed in the easily solubilized fraction but was observed in the poorly soluble protein fraction. These results demonstrated that the 3E1 antibody and the 9D2 antibody caused CADM1 to migrate (internalize) from the easily soluble fraction to the poorly soluble protein fraction.

[0221] (13) Study of Internalization of Anti-CADM1 Antibody The internalization of the anti-CADM1 antibody itself was studied. Specifically, the 3E1 antibody (chicken-mouse chimeric antibody) obtained in Example 1 (1-1) above was labeled with fluorescein to obtain a fluorescein-labeled 3E1 antibody. Subsequently, in a standard mesothelioma cell (NCI-H28) culture system, the fluorescein-labeled 3E1 antibody and the chicken 9D2 antibody obtained in Example 2 (1) above were added to the culture medium at 1 μg / ml each. One, two, and five hours after the addition, fluorescein fluorescence was detected by live cell observation using a confocal laser microscope system (C2+, Nikon Corporation). These results are shown in Figure 23.

[0222] Figure 23 is a photograph showing the results of fluorescence observation of fluorescein. From left to right in Figure 23, the results 1 hour, 2 hours, and 3 hours after addition are shown. As shown in Figure 23, it was found that the 3E1 antibody aggregated on the cell membrane and then migrated into the cell (internalized).

[0223] (14) Study of the efficacy of antibody-drug conjugates based on anti-CADM1 antibodies Figure 24 shows a schematic diagram of an antibody-drug conjugate based on an anti-CADM1 antibody. Specifically, the antibody-drug conjugate (3E1-MMAE) is shown, in which the antibody moiety of the microtubule polymerization inhibitor (MMAE)-conjugated anti-CD30 monoclonal antibody Adcetris (brentuximab vedotin; a therapeutic agent for malignant lymphoma) is replaced with the 3E1 antibody (chicken-mouse chimeric antibody) obtained in Example 1 (1-1). In the 3E1 antibody-MMAE, the 3E1 antibody and MMAE are linked via a cathepsin-cleavable linker.

[0224] The efficacy of the 3E1 antibody-MMAE was investigated. Specifically, 1 μg / ml of each of the 3E1 antibody-MMAE or a control antibody-drug conjugate (control antibody-MMAE) in which the 3E1 antibody was replaced with a control antibody (mouse IgG1) was added to a CADM1-expressing cell culture system (a culture system of the adult T-cell leukemia cell line MT-2, which highly expresses CADM1), and the change in cell number was observed over time. These results are shown in Figure 25. In addition, the cells were observed under a microscope 7 days after addition of the conjugate.

[0225] Figure 25 shows the results of monitoring the cell count of CADM1-expressing cells over time. In Figure 25, the graph shows the change in cell count over time. The upper right-hand column shows a photograph of cells treated with a control antibody and MMAE, while the lower right-hand column shows a photograph of cells treated with a 3E1 antibody (antibody A) and MMAE. As shown in Figure 25, in the control antibody-MMAE group, the cell count increased over time, whereas in the 3E1 antibody-MMAE group, the cell count did not increase after day 3 and began to decrease by day 7. These results demonstrated that MMAE acted intracellularly in the antibody-drug conjugate using the 3E1 antibody. This suggests that the 3E1 antibody is effective as a drug delivery vector. In the results shown in Figure 25, the "P value" of the Student's t-test was less than 0.01, confirming a significant difference between the two groups.

[0226] (15-1) Investigation of the Effectiveness of Anti-CADM1 Antibodies as Intracellular Delivery Vectors for Antibody Drugs The results of Example 4(14) above demonstrated that antibody-drug conjugates based on the 3E1 antibody are effective as drug delivery vectors. Therefore, we next investigated whether the 3E1 antibody could be used to load antibody drugs. Specifically, a h3E1C antibody-mouse IgG1 (Mouse IgG1 kappa Isotype Control, Invitrogen, Cat. No.: 16-4714-82) conjugate was prepared by crosslinking using an amine-reactive crosslinker (e.g., using the Click-&-Go Lys-to-Lys Protein-Protein Conjugation Kit (Click Chemistry Tools)) (Figure 26).

[0227] The 3E1 antibody-mouse IgG1 complex was added to the culture medium of mesothelioma cells (EHMES10) in an amount equivalent to 1 μg of each antibody. The day after the addition, proteins were extracted from the cells (easily soluble and poorly soluble protein fractions) and subjected to Western blotting. For negative controls, the same procedure was used except that the 3E1 antibody (antibody A) was added without cross-linking with mouse IgG1, or the chicken 9D2 antibody obtained in Example 2(1) was also added. These results are shown in Figure 27.

[0228] Figure 27 is a photograph showing the results of Western blotting. In Figure 27, the left vertical axis indicates the type of antibody used for detection, the right vertical axis indicates molecular weight (kDa), and the horizontal axis indicates the type of sample. In Figure 27, the heavy chain released from the complex refers to the heavy chain derived from mouse IgG1 bound to the 3E1 antibody. As shown in Figure 27, the 3E1 antibody-mouse IgG1 complex was found to promote the internalization of CADM1 to the same extent as the 3E1 antibody alone. The 3E1 antibody-mouse IgG1 complex was also detected in the poorly soluble protein fraction.

[0229] (15-2) Investigation of the effectiveness of anti-CADM1 antibodies as intracellular delivery vectors for antibody drugs. The 3E1 antibody-mouse IgG1 complex and the chicken 9D2 antibody obtained in Example 2(1) were added in an amount equivalent to 1 μg of each antibody to the culture medium of mesothelioma cells (EHMES10). 24 hours after the addition, the cells were immunostained. Specifically, mesothelioma cells (EHMES10) were fixed with methanol and then reacted with a mixture of anti-mouse IgG Fc antibody (AffiniPure™ Mouse Anti-Human IgG, Fcγ fragment specific, Jackson ImmunoResearch, Cat. No.: 209-005-098) and anti-human IgG Fc antibody (AffiniPure™ Goat Anti-Mouse IgG, Fcγ fragment specific, Jackson ImmunoResearch, Cat. No.: 115-005-071) at 4°C overnight. Subsequently, the immunostaining was performed using fluorescently labeled secondary antibodies (Alexa Fluor® 594 AffiniPure™ Donkey Anti-Mouse IgG (H+L), Jackson ImmunoResearch, Cat. No. 715-585-150 and Alexa Fluor® 488 AffiniPure™ Donkey Anti-Goat IgG (H+L), Jackson ImmunoResearch, Cat. No. 705-545-003) to detect the localization of the primary antibodies. The immunostaining was then performed using the confocal laser scanning microscope system. The results are shown in Figure 28.

[0230] Figure 28 is a photograph showing the results of immunostaining. In Figure 28, (a) shows the results of immunostaining with anti-mouse IgG Fc antibody, (b) shows the results with anti-human IgG Fc antibody, (c) shows the results with anti-mouse IgG Fc antibody and anti-human IgG Fc antibody, and (d) shows the results of differential interference contrast (DIC). As shown in Figure 28, mouse IgG and human IgG were detected in the form of fine granules within the cells, demonstrating their co-localization. These results suggest that the 3E1 antibody-mouse IgG1 complex was taken up into the cells in its intact form.

[0231] These results suggest that the 3E1 antibody, as an intracellular delivery vector, may be able to efficiently deliver antibody drugs, such as anti-amyloid beta antibodies (Aducanumab) or anti-PD-1 antibodies (Nivolumab), into the intracranial space. Furthermore, the 3E1 antibody is expected to be a vector for intracellular delivery of intracellular antigen-targeting antibodies against tumor intracellular antigens (e.g., KRAS, BRAF, and EGFR kinase domains), which have recently attracted attention as antitumor antibody drugs. When preparing conjugates of the 3E1 antibody with the anti-amyloid beta antibody Aducanumab, the anti-PD-1 antibody Nivolumab, or intracellular tumor antigen-targeting antibodies, the 3E1 antibody and the antibody drug may be linked via a cathepsin-cleavable linker, instead of a crosslink, as in the 3E1 antibody-MMAE conjugate shown in Figure 24.

[0232] (16-1) Study of the Structure of Anti-CADM1 Antibodies for High Probability of CADM1 Internalization The present inventors have found that the anti-CADM1 antibodies 3E1 and 9D2 can internalize CADM1 when added alone. This phenomenon is thought to be initiated by the binding of the 3E1 and 9D2 antibodies to the extracellular domain of CADM1. The present inventors have also found that the simultaneous addition of the 3E1 and 9D2 antibodies dramatically enhances CADM1 internalization. The mechanism behind this can be explained as follows, as shown in Figure 29. As shown in Figure 29(a), CADM1 molecules normally bind to each other to adhere cells to each other, but when the 3E1 antibody (antibody A) and the 9D2 antibody (antibody B) bind to CADM1, the CADM1 molecules are dissociated from each other. As shown in Figure 29(b), the 3E1 antibody is thought to have strong binding affinity to CADM1, and free CADM1 molecules on the cell membrane are attracted to the 3E1 antibody. As a result, a large number of CADM1 molecules associate and aggregate on the cell membrane. In other words, it is speculated that the binding site of the 3E1 antibody to CADM1 is in a favorable position for this action. As shown in Figure 29(c), it is thought that the association and aggregation of CADM1 triggers the internalization of CADM1 (depression of the cell membrane). At this time, the 3E1 antibody, which has strong binding affinity, is also internalized. The 9D2 antibody is also internalized, but the amount internalized is smaller than that of the 3E1 antibody.

[0233] Furthermore, the inventors of the present application have discovered the following: (1) CADM1 molecules exist as cis dimers on the cell membrane and are bound trans (with the molecules facing each other vertically) between adjacent cells. (2) The 9D2 antibody inhibits the trans binding of CADM1 (functions as a neutralizing antibody), so in the presence of anti-CADM1 antibody B, non-trans-bound (free) CADM1 cis dimers are generated on the cell membrane. (3) The 3E1 antibody has a bivalent variable region that strongly binds to the CADM1 extracellular domain, thereby causing the aggregation of CADM1 cis dimers on the cell membrane. (4) When the aggregates of CADM1 molecules reach a certain size, they stimulate internalization, and simultaneously, the 3E1 antibody is internalized.

[0234] Therefore, we investigated the structural requirements (class selectivity) of anti-CADM1 antibodies for CADM1 internalization. Specifically, various cell lines (NCI-H441, NCI-H28, Meso1-CADM1) were cultured in a conventional manner, and 1 μg / ml each of the following antibodies was added to the culture medium: 3E1 antibody and control antibody (human IgM); 3E1 antibody and IgG-type 9D2 antibody (9D2 antibody (G), antibody B (G)); or 3E1 antibody and IgM-type 9D2 antibody (9D2 antibody (M), antibody B (M)). The day after addition, the readily soluble and insoluble protein fractions were extracted and subjected to Western blotting. The class of the 3E1 antibody (antibody A) was IgG. These results are shown in Figure 30.

[0235] Figure 30 is a photograph showing the results of Western blotting. In Figure 30, the left vertical axis indicates the type of antibody used for detection, the right vertical axis indicates molecular weight (kDa), and the horizontal axis indicates the type of sample. As shown in Figure 30, when the 9D2 antibody was IgM, CADM1 internalization was very efficient, but when the 9D2 antibody was IgG, CADM1 internalization was significantly lower. The 9D2 antibody (M) inhibits trans binding between CADM1 molecules (has neutralizing activity), whereas the 9D2 antibody (G) does not have neutralizing activity. Therefore, neutralizing activity was identified as a functional requirement for the 9D2 antibody to promote CADM1 internalization. When the structure of the antibody constant region is of the IgM class (isotype), IgM forms multimers (pentamers or hexamers). Therefore, it is thought that a mechanism for promoting internalization is formed by the attachment of 9D2 antibody multimers to the CADM1 aggregates in the aforementioned (4).

[0236] (16-2) Study of Anti-CADM1 Antibody Structure for High Probability of CADM1 Internalization We further investigated the structural requirements of anti-CADM1 antibodies for CADM1 internalization. Specifically, mesothelioma cells (Meso1-CADM1) were cultured in a conventional manner, and 1 μg / ml each of the following was added to the culture medium: an IgG control antibody (human IgG) and an IgM control antibody (human IgM) (control G + M), an IgM 3E1 antibody (3E1 antibody (M)), an IgM 3E1 antibody and an IgM 9D2 antibody (3E1 antibody (M) + 9D2 antibody (M)), or an IgG 3E1 antibody and an IgM 3E1 antibody (3E1 antibody (G) + 3E1 antibody (M)). The day after addition, the readily soluble and insoluble protein fractions were extracted and subjected to Western blotting. The results are shown in Figure 31.

[0237] Figure 31 is a photograph showing the results of Western blotting. In Figure 31, the left vertical axis indicates the type of antibody used for detection, the right vertical axis indicates molecular weight (kDa), and the horizontal axis indicates the type of sample. In Figure 31, nonspecific bands refer to bands that arise when an antibody reacts with some molecule other than its native antigen, CADM1. As shown in Figure 31, in the groups to which the IgM 3E1 antibody and the IgM 9D2 antibody were added, CADM1 and 3E1 antibodies (antibodies (heavy chains) in the figure) were detected in the poorly soluble fraction. This indicates that internalization of CADM1 and anti-CADM1 antibody A had occurred. On the other hand, in the groups to which the IgG 3E1 antibody and the IgM 3E1 antibody were added, CADM1 and 3E1 antibodies were not detected in the poorly soluble fraction. This indicates that internalization of CADM1 and anti-CADM1 antibody A had not occurred. As mentioned above, the IgM 9D2 antibody has neutralizing activity against CADM1 trans binding, whereas the IgM 3E1 antibody does not. Therefore, it was found that the neutralizing activity is a requirement for the 9D2 antibody to promote the internalization of CADM1 and 3E1 antibodies.

[0238] (16-3) Study of the Structure of Anti-CADM1 Antibodies for High-Probability Internalization of CADM1 The simultaneous addition of 3E1 (Antibody A) and 9D2 (Antibody B) antibodies was investigated to improve the efficiency of CADM1 internalization. Specifically, various cells (dorsal root ganglion cells, RGC-5, and NCI-H441) were cultured in a conventional manner, and the 3E1 antibody (chicken-mouse chimeric antibody) obtained in Example 1 (1-1) and the chicken 9D2 antibody obtained in Example 2 (1) (each at 1 μg / ml), or the control antibody chicken IgY (2 μg / ml, Normal chicken IgY control, R&D, Cat. No.: AB-101-C) were added to the culture medium. The day after the addition, a poorly soluble protein fraction was extracted from the cells and subjected to Western blotting together with the culture medium protein sample. The results are shown in Figure 32.

[0239] Figure 33 is a photograph showing the results of Western blotting. In Figure 33, the vertical axis indicates the type of antibody used for detection, and the horizontal axis indicates the type of sample. Taking into account the dilution rate of the protein sample, it is estimated that nearly half of the 3E1 antibody (heavy chain) added to the culture medium was internalized into the cells. In other words, when the 3E1 antibody and the 9D2 antibody were added simultaneously to a standard culture system of CADM1-expressing cells, CADM1 was internalized with very high efficiency, and the 3E1 antibody was also internalized with high efficiency.

[0240] (16-4) Study of the Structure of Anti-CADM1 Antibodies for High-Probability Internalization of CADM1 Furthermore, we investigated how the internalization efficiency varied depending on the concentration and quantitative ratio of the 3E1 antibody (antibody A) and the 9D2 antibody (antibody B). Specifically, various cells (NCI-H441, HEC1B) were cultured in a conventional manner, and the 3E1 antibody (chicken-mouse chimeric antibody) obtained in Example 1 (1-1) above, the chicken 9D2 antibody obtained in Example 2 (1) above, or a control antibody (chicken IgY, Normal chicken IgY control, R&D, Cat. No.: AB-101-C) was added to the culture medium at various concentrations. The day after the addition, the readily soluble and poorly soluble protein fractions were extracted and subjected to Western blotting. These results are shown in Figure 33.

[0241] Figure 33 is a photograph showing the results of Western blotting. In Figure 33, the left vertical axis indicates the type of antibody used for detection, the right vertical axis indicates molecular weight (kDa), and the horizontal axis indicates the type of sample. As shown in Figure 33, when the quantitative ratio of 3E1 antibody:9D2 antibody was fixed at 1:1 and the total concentration of 3E1 antibody and 9D2 antibody was varied in the range of 10 to 0.01 μg / ml, the internalization efficiency decreased in a concentration-dependent manner, and almost no internalization occurred at 0.01 μg / ml.

[0242] (16-5) Study of the Structure of Anti-CADM1 Antibodies for High Probability of CADM1 Internalization We investigated how the internalization efficiency changed when the concentration of the 3E1 antibody (antibody A) was fixed and the concentration of the 9D2 antibody (antibody B) was varied. Specifically, NCI-H441 lung epithelial cells were cultured in a conventional manner, and the 3E1 antibody (chicken-mouse chimeric antibody) obtained in Example 1 (1-1), the chicken 9D2 antibody obtained in Example 2 (1), or the control antibody (chicken IgY) was added to the culture medium at various concentrations. The day after the addition, the readily soluble fraction was extracted and subjected to Western blotting. The results are shown in Figure 34.

[0243] Figure 34 is a photograph showing the results of Western blotting. In Figure 34, the left vertical axis indicates the type of antibody used for detection, the right vertical axis indicates molecular weight (kDa), and the horizontal axis indicates the type of sample. As shown in Figure 34, the concentration of the 3E1 antibody was fixed at 5 μg / ml, and the quantitative ratio of the 9D2 antibody to the 3E1 antibody was varied in the range of 1:1 to 1:0.01. As the quantitative ratio of the 9D2 antibody to the 3E1 antibody decreased, the efficiency of CADM1 internalization also decreased, and at a quantitative ratio of the 9D2 antibody to the 3E1 antibody of 1:0.01, almost no CADM1 internalization occurred.

[0244] Example 5 It was confirmed that anti-CADM1 antibodies can suppress pain and that the anti-CADM1 antibodies accumulate in peripheral nerves.

[0245] (1) Pain-suppressing function of anti-CADM1 antibodies. The pain-suppressing function of anti-CADM1 antibodies was examined using a mouse pain model. First, 3E1 antibody (chicken-mouse chimeric antibody, antibody A) or a control antibody (mouse IgG1) was subcutaneously administered to the soles of B57BL / 6 mice (n = 6 per group). Each antibody was administered at a dose of 20 μg into both the left and right soles. A negative control (n = 6) was also performed in the same manner, except that phosphate buffered saline (PBS) alone was administered subcutaneously instead of the antibody. Six hours after administration of the antibody or PBS, formalin was administered subcutaneously into both the left and right soles. Immediately after administration, mouse behavior was videotaped. The videotapes were then observed for the first 5 minutes (0-5 minutes), the next 5 minutes (5-10 minutes), and then at 10-minute intervals (10-20 minutes, 20-30 minutes, and 30-40 minutes). The sum of the duration of pain-related behaviors within each time period was calculated. The pain-related behaviors were licking, biting, lifting, withdrawing, and shaking of the paw by the mice. The results are shown in Figure 35.

[0246] Figure 35 is a graph showing the duration of pain-related behavior. In Figure 35, the horizontal axis represents the time after formalin administration, and the vertical axis represents the total duration of pain-related behavior. In Figure 35, the average duration of pain-related behavior in each time segment is plotted together with the standard deviation. As shown in Figure 35, the 3E1 antibody administration group had the shortest total duration of pain-related behavior in all time segments.

[0247] In this pain model, the first 10 minutes after formalin administration are called the chemical pain phase (Phase I), and the following 30 minutes are called the inflammatory pain phase (Phase II). Therefore, the duration of pain-related behaviors during Phase I and Phase II was compared between each group. The results are shown in Figure 36.

[0248] Figure 36 is a graph showing the duration of pain-related behavior in each phase. In Figure 36, the horizontal axis represents each administration group, and the vertical axis represents the sum of the durations of pain-related behavior. Figure 36 also shows the mean and standard deviation for each group, along with the P value (Student t-test) for intergroup comparisons. As shown in Figure 36, the duration of pain-related behavior was shortest in the 3E1 antibody-administered group in both Phase I and Phase II. These results demonstrate that the 3E1 antibody can suppress both chemical injury pain and inflammatory pain, two different types of pain.

[0249] Next, the sum of the time spent exhibiting pain-related behavior was calculated in the same manner, except that the 45TG23 antibody was used in addition to the 3E1 antibody. The results are shown in Figure 37.

[0250] Figure 37 is a graph showing the duration of pain-related behavior. In Figure 37, the horizontal axis represents the time after formalin administration, and the vertical axis represents the total duration of pain-related behavior. Also, in Figure 37, the average duration of pain-related behavior in each time segment is plotted along with the standard deviation. As shown in Figure 37, the 45TG23 antibody-administered group, like the 3E1C antibody (antibody A)-administered group, exhibited a suppression of the total duration of pain-related behavior in all time segments. Also, as shown in Figure 37, the 45TG23 antibody-administered group exhibited a suppression of the total duration of pain-related behavior in both phases I and II. These findings demonstrate that anti-CADM1 antibodies can suppress both chemical injury pain and inflammatory pain, two different types of pain.

[0251] (2) Accumulation of anti-CADM1 antibodies in peripheral nerves To investigate the mechanism of action of anti-CADM1 antibodies in suppressing pain, we investigated whether anti-CADM1 antibodies accumulate in peripheral nerves associated with pain. Specifically, 20 μg of 3E1 antibody (chicken IgY antibody) was administered near the epidural space at the first / second lumbar intervertebral space of the back of C57BL / 6 mice, as indicated by the arrow X in Figure 38 . Five hours after administration, tissues from the administration site were collected, and tissue sections were prepared. After HE staining, the tissue sections were immunostained to examine the localization of the 3E1 antibody using HRP-labeled anti-chicken IgY antibody and a substrate. The results are shown in Figure 39 .

[0252] Figure 39 is a photograph showing a tissue section of the administration site. As indicated by the triangle in Figure 39, the anti-CADM1 antibody was specifically and strongly accumulated in the dorsal root ganglion.

[0253] (3) Accumulation of anti-CADM1 antibodies in peripheral nerves via different administration routes. We investigated whether different administration routes result in accumulation of anti-CADM1 antibodies in peripheral nerves. Specifically, the 3E1 antibody (chicken-mouse chimeric antibody, antibody A) and a control antibody (mouse IgG1) were labeled with indocyanine green (ICG), and the labeled antibodies were administered via the tail vein of C57BL / 6 mice (5 mg / kg body weight). ICG fluorescence was detected in each mouse using the Pearl Trilogy system 1 and 28 hours after administration. The results are shown in Figure 40.

[0254] Figure 40 is a photograph of a fluorescence image showing the localization of the anti-CADM1 antibody. In Figure 40, the upper row shows a fluorescence image 1 hour after administration, and the lower row shows a fluorescence image 28 hours after administration. In Figure 40, each photograph shows, from left to right, the results of the 3E1 antibody-administered group and the control antibody-administered group. As shown in Figure 40, accumulation of ICG fluorescence was observed in the dorsal skin, palms, and feet of mice in the 3E1 antibody-administered group 1 and 28 hours after administration.

[0255] Next, the location of ICG fluorescence accumulation was confirmed by preparing frozen sections of mouse tissues 28 hours after administration and observing them under a microscope. Specifically, tissues including the dorsal skin and plantar skin were collected and then frozen sections were prepared. The obtained frozen sections were observed under a phase-contrast microscope and a fluorescence microscope to examine the localization in each tissue. The results are shown in Figure 41.

[0256] Figure 41 is a photograph showing fluorescence and phase-contrast images of ICG in peripheral tissues. In Figure 41, each photograph shows, from the top to bottom, a merged image of phase-contrast and fluorescence images of dorsal skin, a fluorescence image of dorsal skin, and a merged image of phase-contrast and fluorescence images of plantar skin. As indicated by the triangles in Figure 41, ICG fluorescence of the 3E1 antibody was detected on peripheral nerve fibers in the dermis or subcutaneous fat tissue of the dorsal and plantar regions. In other words, the intravenously administered 3E1 antibody was specifically localized to peripheral nerves for a long period of time in a wide range of tissues, such as the dorsal skin and extremities.

[0257] From the above results, it was considered that anti-CADM1 antibodies widely accumulate in peripheral nerve fibers and inhibit the transmission of nerve stimuli such as pain and pruritus, thereby exerting effects similar to those of local anesthetics (e.g., dulling pain and pruritus sensation). In other words, it was considered that anti-CADM1 antibodies dull peripheral nerve hypersensitivity, such as peripheral pain and pruritus sensation, and can be used to suppress peripheral pain and pruritus. Therefore, it is expected that anti-CADM1 antibodies can be used as local anesthetics and pharmaceuticals for systemic relief of peripheral neuralgia. Furthermore, it is expected that routes of administration for localization of the anti-CADM1 antibodies that can act on peripheral nerves, such as the affected area, the epidural space, or intravenous administration, can be used.

[0258] Furthermore, as mentioned above, anti-CADM1 antibodies can efficiently induce cellular internalization, and therefore can be used as drug delivery vectors for peripheral nerves, and are expected to be used as vectors for neuroprotective drugs, nucleic acid drugs, and the like. Furthermore, if an antibody-drug conjugate loaded with a local anesthetic (procaine, etc.) is prepared and administered subcutaneously or epidurally, it is conceivable that the effects of the anesthetic may be exerted for a very long period of time. In other words, anti-CADM1 antibodies are expected to become long-acting local anesthetics.

[0259] [Example 6] It was confirmed that the anti-tumor activity was enhanced by combining with an anti-CADM1 antibody.

[0260] Mouse melanoma B16 cells (3 × 10 6) was suspended in 50% Matrigel (BD Bioscience) and injected subcutaneously into the flanks of 6- to 10-week-old C57BL / 6 mice (Japan SLC, Shizuoka Prefecture, Japan) (six female mice). 3 Once tumor size reached 800 mm or greater, mouse IgG (mIgG)-MMAE conjugate plus mouse IgM (circle in the figure) or chicken-mouse chimeric 3E1 (cm3E1)-MMAE conjugate plus chicken-mouse chimeric 9D2 (cm9D2) (▲ in the figure) (each 0.1 mg / mL, volume equal to the tumor size) was locally injected twice a week. 3 The test was terminated when the cells reached 0.05 or became undetectable by eye (16 to 25 days after transplantation). The results are shown in Figure 42.

[0261] Figure 42 shows a graph and photographs showing tumor size. In Figure 42, (a) shows the number of days after tumor implantation, and (b) shows a graph showing tumor weight at the end of the test and a photograph showing a representative example of tumor tissue. In Figure 42(a), the horizontal axis shows the number of days after tumor implantation, and the vertical axis shows tumor size. As shown in Figure 42(a), the group receiving local administration of the chicken-mouse chimera 3E1-MMAE conjugate plus the chicken-mouse chimera 9D2 significantly suppressed the growth of subcutaneous B16 melanoma in mice compared to the group receiving administration of the mouse IgG-MMAE conjugate plus mouse IgM. Furthermore, as shown in Figure 42(b), tumor burden was dramatically reduced (92.7%) (tumor weight: 0.064 ± 0.028 g in the group treated with chicken-mouse chimera 3E1-MMAE conjugate + chicken-mouse chimera 9D2, 0.87 ± 0.15 g in the group treated with mouse IgG-MMAE conjugate + mouse IgM; p = 0.00014 by unpaired two-tailed Student's t-test). These results confirmed that the 3E1-MMAE conjugate exhibits antitumor activity against CADM1-expressing tumors. Furthermore, the antitumor activity was enhanced by combining the 3E1-MMAE conjugate with the 9D2 antibody compared to when the 3E1-MMAE conjugate was used alone, suggesting that the antitumor activity can be enhanced by combining the conjugate with an anti-CADM1 antibody.

[0262] Example 7 It was confirmed that anti-CADM antibodies can be used to prevent or treat type 1 diabetes.

[0263] In a type 1 diabetes insulitis model, it was confirmed that anti-CADM1 antibody exhibits a protective effect on β cells. Mouse β cell line Min6 cells were suspended in medium and then cultured at 5 × 10 ４ The cells were seeded at 200 μL / well in a 96-well plate (Elplasia plate 96 well, Corning, Cat. No.: 4442). The medium used was D-MEM medium (high glucose, phenol red-free; FUJIFILM, Cat. No.: 040-30095) containing 10% fetal bovine serum (FBS). After seeding, the cells were incubated at 37°C and 5% CO ２ The cells were cultured under these conditions for 2 days to form spheroids.

[0264] The resulting Min6 cell spheroids were pretreated with a control antibody, human IgM (ChromPure Human IgM (myeloma), whole molecule; JacksonImmunoResearch, 009-000-012), or the humanized 9D2 antibody clone H2L2, each at 1 μg / mL.

[0265] Jurkat cells, a human T-cell leukemia cell line, were prepared as cells for inducing human β-cell damage. Specifically, Jurkat cells were stimulated with LPS (final concentration 100 ng / mL) (Invitrogen, Cat. No.: 00-4976-93) to induce cytotoxic activity. Two hours after the stimulation, the activated Jurkat cells were collected, washed with the D-MEM medium, and diluted to 5 × 10 ４ The concentration was adjusted to cells / 10 μL.

[0266] To each well containing the pretreated Min6 cell spheroids, 10 μL (5×10 ４Cells) were seeded and co-cultured for one day. As a control, each well was washed with the D-MEM medium to remove the antibody, and then cultured in the same manner in the presence of the D-MEM medium. After the culture, 100 μL of the supernatant was collected and transferred to a 96-well plate for measurement. Cytotoxicity LDH Assay Kit-WST (DOJINDO, Cat. No.: CK12) was used to measure cytotoxic activity. The activity value of the group without control antibody (anti-human IgM) pretreatment or Jurkat co-culture was set at 1, and the relative activity value of each group was calculated, followed by calculation of the mean and standard deviation. The results are shown in Figure 43.

[0267] Figure 43 is a graph showing the relative values ​​of cytotoxic activity. In Figure 43, the horizontal axis indicates the type of sample group, and the vertical axis indicates the relative values ​​of cytotoxic activity. As shown in Figure 43, when β cells were cultured with Jurkat cells, β cell cytotoxicity was induced. In contrast, in the group to which the 9D2 antibody was added, β cell damage caused by Jurkat cells was suppressed. These results demonstrate that anti-CADM1 antibodies can suppress β cell damage caused by T cells in type 1 diabetic insulitis.

[0268] [Example 8] It was confirmed that an anti-CADM1 antibody can improve dermatitis and reduce scratching behavior time in atopic dermatitis.

[0269] (1) Examination of Healing in an Atopic Dermatitis Model Using Anti-CADM1 Antibody The therapeutic effect of an anti-CADM1 antibody was examined using an atopic dermatitis model. Figure 44 shows an overview of the preparation of an atopic dermatitis model and antibody administration. As shown in Figure 44, the atopic dermatitis model was prepared and the antibody was administered via the tail vein in the same manner as in Example 4(2) above, except that the 3E1 antibody (chicken-mouse chimeric antibody) or control antibody was injected three times every other day, on the day of grouping, the day after that, and the day after that, at a dose of 5 mg / kg body weight (n = 6 per group).

[0270] (2) Evaluation of Dermatitis Score Regarding the severity of dermatitis, various symptoms were scored on the day of grouping (day 0) and a predetermined number of days after the day of grouping (day 11 or 15) in the same manner as in Example 4(2) above ( FIG. 44 ), and the sum of scores A to D for each mouse was used as the dermatitis score for each individual. Furthermore, the relative dermatitis score was calculated by dividing the sum of the dermatitis scores for each mouse by the sum of the dermatitis scores for each mouse on the day of grouping. Statistical analysis between the 3E1 antibody-administered group and the control antibody-administered group was performed using Student's t test. The average relative scores for each group are shown in FIG. 45 .

[0271] Figure 45 is a graph showing the relative dermatitis scores in each group. In Figure 45, the horizontal axis represents the number of days after grouping, and the vertical axis represents the relative dermatitis scores. As shown in Figure 45, on days 11 and 15 after grouping, the 3E1 antibody-administered group showed a significant improvement in dermatitis score compared to the control antibody-administered group (p < 0.05).

[0272] (3) Evaluation of Itching Behavior We investigated whether administration of anti-CADM1 antibodies inhibits scratching behavior, an indicator of itching. Specifically, the behavior of each mouse from Example 8(1) was videotaped for 30 minutes on the day of grouping (first time) and 5 days after grouping (second time). Using the obtained video, the time spent by each mouse engaged in scratching behavior was calculated. Then, for each mouse, the time spent on scratching behavior on the day of grouping (first time) was defined as 1, and the relative value of the time spent on scratching behavior on the second time was calculated. Statistical analysis between the 3E1 antibody-administered group and the control antibody-administered group was performed using the Mann-Whitney U test. The average values ​​for each group are shown in Figure 46.

[0273] Figure 46 is a graph showing the relative values ​​of the scratching behavior time in each group. In Figure 46, the horizontal axis represents each administration group and the number of behavioral analyses, and the vertical axis represents the relative values ​​of the scratching behavior time. As shown in Figure 46, in the second behavioral analysis, the scratching behavior time was significantly reduced in the 3E1 antibody administration group.

[0274] [Example 9] It was confirmed that anti-CADM1 antibodies can prevent type 1 diabetes.

[0275] (1) Evaluation of Diabetes Prevention The ability of anti-CADM1 antibodies to suppress the onset of type 1 diabetes was examined using NOD mice. First, the humanized 9D2 antibody (with H2L2 and J chain) obtained in Example 2 (2-2) and the control antibody (human IgM) were diluted to 0.5 mg / mL in PBS. The NOD mice were divided into two groups: one group (four mice) administered with the humanized 9D2 antibody and one group (four mice) administered with the control antibody. The NOD mice were housed in cages of three mice (two NOD mice and one dummy mouse), including a dummy mouse. Administration of the 9D2 antibody or control antibody began at 14 weeks of age. Prior to administration, each NOD mouse was confirmed to be negative for glucose in urine. The antibodies were administered intraperitoneally to NOD mice at 1 mg / kg body weight once a week for a total of nine doses. Before each antibody administration, mice were weighed and their urine glucose levels were checked. If the urine glucose level was ± or higher, the blood glucose levels of the mice were measured. If the blood glucose level of the mice was 200 mg / dL or higher, the mice were evaluated as having type 1 diabetes, and the proportion of mice without type 1 diabetes among all mice in each group was calculated. Statistical analysis of the differences between the 9D2 antibody-administered group and the control antibody-administered group was performed using the log-rank test. These results are shown in Figure 47.

[0276] Figure 47 is a graph showing the proportion of individuals who have not developed type 1 diabetes. In Figure 47, the horizontal axis represents the age of the mice in weeks, and the vertical axis represents the proportion of individuals who have not developed type 1 diabetes. As shown in Figure 47, diabetes developed over time in the control group. In contrast, the onset of type 1 diabetes was significantly suppressed in all NOD mice in the humanized 9D2 antibody-administered group (p = 4.37 × 10 -5 ) Therefore, it was found that anti-CADM1 antibodies can suppress the onset of type 1 diabetes.

[0277] (2) Evaluation of Glucose Tolerance A glucose tolerance test was performed to examine whether anti-CADM1 antibodies could improve glucose tolerance. Specifically, humanized 9D2 (with H2L2 and J chain) was prepared at 0.5 mg / mL in PBS, as described in Example 9(1). PBS was used as a control. NOD mice were divided into a humanized 9D2 antibody-treated group (3 mice) and a control group (3 mice). The NOD mice were housed in cages of 3 mice each, and from 13 weeks of age, the 9D2 antibody or PBS was administered. The antibody was administered to the NOD mice via the tail vein at 10 mg / kg mouse body weight once a week for a total of three doses. A glucose tolerance test was performed the week after the final administration. The mice were fasted for at least 10 hours from the day before the glucose tolerance test, and urine glucose was confirmed to be negative before the test began. After this confirmation, glucose solution was administered intraperitoneally at 2 mg / g mouse body weight. Blood was collected from the tail of the NOD mice at 0 (just before administration of glucose solution), 10, 30, 60, and 120 minutes after administration, and blood glucose levels were measured. Statistical analysis between the 9D2 antibody-administered group and the control group was performed using Student's t test. The results are shown in Figure 48.

[0278] Figure 48 is a graph showing blood glucose levels. In Figure 48, the horizontal axis represents the time after glucose solution administration, and the vertical axis represents blood glucose levels. As shown in Figure 48, the 9D2 antibody administration group had lower blood glucose levels than the control group at all timings. In particular, the 9D2 antibody administration group showed a significant decrease in blood glucose levels 30 and 60 minutes after glucose solution administration (p < 0.05). Therefore, it was found that anti-CADM1 antibodies can improve glucose tolerance.

[0279] Although the present disclosure has been described above with reference to the embodiments and examples, the present disclosure is not limited to the above-described embodiments and examples. Various modifications that can be understood by those skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0280] This application claims priority based on Japanese Patent Application No. 2024-090930, filed on June 4, 2024, the disclosure of which is incorporated herein in its entirety by reference.

[0281] <Appendix> Some or all of the above embodiments and examples can be described as, but are not limited to, the following appendixes. (Appendix 1) <Pharmaceutical composition for use in treating a CADM1-related disease> (Appendix 2) A pharmaceutical composition for use in treating a CADM1-related disease, comprising an antibody against cell adhesion molecule 1 (CADM1) or an antigen-binding fragment thereof. (Appendix 3) The pharmaceutical composition according to Appendix 1, wherein the anti-CADM1 antibody or antigen-binding fragment thereof binds to an epitope comprising amino acid residues at positions 311, 325, and 326 in CADM1. (Appendix 4) The pharmaceutical composition according to Appendix 2, wherein the anti-CADM1 antibody or antigen-binding fragment thereof binds to an epitope comprising amino acid residues at positions 311, 312, 325, and 326 in CADM1. (Supplementary Note 5) The anti-CADM1 antibody or antigen-binding fragment thereof has heavy chain CDR1 to 3 and light chain CDR1 to 3, each of which has the amino acid sequence of heavy chain CDR1 to 3 and light chain CDR1 to 3 of 3E1C antibody, 3E1A antibody, 3E1D antibody, 3E1Y antibody, 3E1V antibody, 3E1E antibody, 3E1F antibody, 3E1G antibody, 3E1H antibody, 3E1I antibody, 3E1K antibody, 3E1L antibody, 3E1M antibody, 3E1N antibody, 3E1P antibody, 3E1Q antibody, 3E1R antibody, 3E1S antibody, 3E1W antibody, 3E1T antibody, h3E1A antibody, h3E1F antibody, or h3E1V antibody shown in Table 1A to B; or The pharmaceutical composition according to any one of Appendices 1 to 3, comprising the amino acid sequences of heavy chain CDR1 to 3 and light chain CDR1 to 3 of the 45TG2 antibody, 45TG3 antibody, 45TG23 antibody, 45TG24 antibody, 45XL7 antibody, 45XL9 antibody, 45XL11 antibody, 45XL14 antibody, 45XL34 antibody, 45XL45 antibody, 43TG47 antibody, 43XL1 antibody, 43XL4 antibody, 43XL17 antibody, 1-1 antibody, 1-2 antibody, 1-3 antibody, 1-4 antibody, 2-1 antibody, 2-2 antibody, 2-3 antibody, 2-4 antibody, 3-1 antibody, 3-2 antibody, 3-3 antibody, 3-4 antibody, 4-1 antibody, 4-2 antibody, 4-3 antibody, or 4-4 antibody, as shown in Tables 2A to 2C below.(Supplementary Note 6) The anti-CADM1 antibody or antigen-binding fragment thereof has a heavy chain variable region and a light chain variable region each having the amino acid sequence of the heavy chain and the light chain of the 3E1C antibody, the 3E1A antibody, the 3E1D antibody, the 3E1Y antibody, the 3E1V antibody, the 3E1E antibody, the 3E1F antibody, the 3E1G antibody, the 3E1H antibody, the 3E1I antibody, the 3E1K antibody, the 3E1L antibody, the 3E1M antibody, the 3E1N antibody, the 3E1P antibody, the 3E1Q antibody, the 3E1R antibody, the 3E1S antibody, the 3E1W antibody, the 3E1T antibody, the h3E1A antibody, the h3E1F antibody, or the h3E1V antibody shown in Table 1A to B; or The pharmaceutical composition according to any one of Appendix 1 to 4, comprising the amino acid sequences of the heavy and light chains of 45TG2 antibody, 45TG3 antibody, 45TG23 antibody, 45TG24 antibody, 45XL7 antibody, 45XL9 antibody, 45XL11 antibody, 45XL14 antibody, 45XL34 antibody, 45XL45 antibody, 43TG47 antibody, 43XL1 antibody, 43XL4 antibody, 43XL17 antibody, 1-1 antibody, 1-2 antibody, 1-3 antibody, 1-4 antibody, 2-1 antibody, 2-2 antibody, 2-3 antibody, 2-4 antibody, 3-1 antibody, 3-2 antibody, 3-3 antibody, 3-4 antibody, 4-1 antibody, 4-2 antibody, 4-3 antibody, or 4-4 antibody shown in Table 2A to C. (Supplementary Note 7) The pharmaceutical composition according to Supplementary Note 1, wherein the anti-CADM1 antibody or antigen-binding fragment thereof has heavy chain CDR1 to 3 and light chain CDR1 to 3 comprising the amino acid sequences of heavy chain CDR1 to 3 and light chain CDR1 to 3 of 9D2 antibody, H1L1 antibody, H1L2 antibody, H1L3 antibody, H1LA1 antibody, H1LA2 antibody, H2L1 antibody, H2L2 antibody, H2L3 antibody, H2LA1 antibody, H2LA2 antibody, H3L1 antibody, H3L2 antibody, H3LA1 antibody, or H3LA2 antibody, respectively, as shown in Tables 3A and 3B. (Appendix 8) The pharmaceutical composition according to Appendix 1 or 6, wherein the anti-CADM1 antibody or antigen-binding fragment thereof has a heavy chain variable region and a light chain variable region comprising the amino acid sequences of heavy chain CDR1 to 3 and light chain CDR1 to 3 of 9D2 antibody, H1L1 antibody, H1L2 antibody, H1L3 antibody, H1LA1 antibody, H1LA2 antibody, H2L1 antibody, H2L2 antibody, H2L3 antibody, H2LA1 antibody, H2LA2 antibody, H3L1 antibody, H3L2 antibody, H3LA1 antibody, or H3LA2 antibody, respectively, shown in Tables 3A to 3B.(Appendix 9) The pharmaceutical composition according to Appendices 6 or 7, wherein the antibody is an IgM antibody. (Appendix 10) The pharmaceutical composition according to any of Appendices 1 to 8, wherein the antibody is a monoclonal antibody. (Appendix 11) The pharmaceutical composition according to any of Appendices 1 to 9, wherein the antibody is a humanized antibody. (Appendix 12) The pharmaceutical composition according to any of Appendices 1 to 10, comprising an antibody-drug conjugate comprising the antibody against CADM1 or its antigen-binding fragment and a drug. (Appendix 13) The pharmaceutical composition according to any of Appendices 1 to 11, wherein the CADM1-related disease is selected from the group consisting of allergic diseases, neurological diseases, bone diseases, type 1 diabetes, peripheral neuralgia, and pruritus. (Appendix 14) The pharmaceutical composition according to Appendices 12, wherein the allergic disease is atopic dermatitis. (Appendix 15) The pharmaceutical composition according to Appendices 12, wherein the neurological disease is selected from the group consisting of Alzheimer's disease, stroke, epilepsy, Parkinson's disease, headache, and demyelinating neurological disease. (Appendix 16) The pharmaceutical composition according to Appendix 12, wherein the bone disease is selected from the group consisting of osteoporosis, intractable fractures, and intraosseous nonunions. (Appendix 17) The pharmaceutical composition according to Appendix 12, wherein the peripheral neuralgia is selected from the group consisting of traumatic pain, chemical traumatic pain, and inflammatory pain. <Composition for nasal intracranial delivery> (Appendix 18) A composition for use in intracranial delivery by nasal administration, comprising an anti-CADM1 antibody or its antigen-binding fragment and a drug. (Appendix 19) The composition according to Appendix 17, wherein the anti-CADM1 antibody or its antigen-binding fragment binds to an epitope comprising amino acid residues 311, 325, and 326 in CADM1. (Appendix 20) The composition of Appendices 18, wherein the anti-CADM1 antibody or antigen-binding fragment thereof binds to an epitope comprising amino acid residues at positions 311, 312, 325, and 326 in CADM1.(Supplementary Note 21) The anti-CADM1 antibody or antigen-binding fragment thereof has heavy chain CDR1 to 3 and light chain CDR1 to 3, each of which has the amino acid sequence of heavy chain CDR1 to 3 and light chain CDR1 to 3 of 3E1C antibody, 3E1A antibody, 3E1D antibody, 3E1Y antibody, 3E1V antibody, 3E1E antibody, 3E1F antibody, 3E1G antibody, 3E1H antibody, 3E1I antibody, 3E1K antibody, 3E1L antibody, 3E1M antibody, 3E1N antibody, 3E1P antibody, 3E1Q antibody, 3E1R antibody, 3E1S antibody, 3E1W antibody, 3E1T antibody, h3E1A antibody, h3E1F antibody, or h3E1V antibody shown in Table 1A to B; or The composition according to any of Appendices 17 to 19, comprising the amino acid sequences of heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3 of the 45TG2 antibody, 45TG3 antibody, 45TG23 antibody, 45TG24 antibody, 45XL7 antibody, 45XL9 antibody, 45XL11 antibody, 45XL14 antibody, 45XL34 antibody, 45XL45 antibody, 43TG47 antibody, 43XL1 antibody, 43XL4 antibody, 43XL17 antibody, 1-1 antibody, 1-2 antibody, 1-3 antibody, 1-4 antibody, 2-1 antibody, 2-2 antibody, 2-3 antibody, 2-4 antibody, 3-1 antibody, 3-2 antibody, 3-3 antibody, 3-4 antibody, 4-1 antibody, 4-2 antibody, 4-3 antibody, or 4-4 antibody shown in Table 2A to C.(Supplementary Note 22) The anti-CADM1 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region and the light chain variable region have the heavy chain and light chain amino acid sequences of the 3E1C antibody, 3E1A antibody, 3E1D antibody, 3E1Y antibody, 3E1V antibody, 3E1E antibody, 3E1F antibody, 3E1G antibody, 3E1H antibody, 3E1I antibody, 3E1K antibody, 3E1L antibody, 3E1M antibody, 3E1N antibody, 3E1P antibody, 3E1Q antibody, 3E1R antibody, 3E1S antibody, 3E1W antibody, 3E1T antibody, h3E1A antibody, h3E1F antibody, or h3E1V antibody shown in Table 1A to B; or The composition according to any of Appendices 17 to 20, comprising the amino acid sequences of the heavy and light chains of the 45TG2 antibody, 45TG3 antibody, 45TG23 antibody, 45TG24 antibody, 45XL7 antibody, 45XL9 antibody, 45XL11 antibody, 45XL14 antibody, 45XL34 antibody, 45XL45 antibody, 43TG47 antibody, 43XL1 antibody, 43XL4 antibody, 43XL17 antibody, 1-1 antibody, 1-2 antibody, 1-3 antibody, 1-4 antibody, 2-1 antibody, 2-2 antibody, 2-3 antibody, 2-4 antibody, 3-1 antibody, 3-2 antibody, 3-3 antibody, 3-4 antibody, 4-1 antibody, 4-2 antibody, 4-3 antibody, or 4-4 antibody shown in Table 2A to C. (Appendix 23) The composition according to Appendix 17, wherein the anti-CADM1 antibody or antigen-binding fragment thereof has heavy chain CDR1 to 3 and light chain CDR1 to 3 comprising the amino acid sequences of heavy chain CDR1 to 3 and light chain CDR1 to 3 of 9D2 antibody, H1L1 antibody, H1L2 antibody, H1L3 antibody, H1LA1 antibody, H1LA2 antibody, H2L1 antibody, H2L2 antibody, H2L3 antibody, H2LA1 antibody, H2LA2 antibody, H3L1 antibody, H3L2 antibody, H3LA1 antibody, or H3LA2 antibody, respectively, as shown in Tables 3A and 3B. (Appendix 24) The composition according to Appendix 17 or 22, wherein the anti-CADM1 antibody or antigen-binding fragment thereof has a heavy chain variable region and a light chain variable region comprising the amino acid sequences of heavy chain CDR1 to 3 and light chain CDR1 to 3 of 9D2 antibody, H1L1 antibody, H1L2 antibody, H1L3 antibody, H1LA1 antibody, H1LA2 antibody, H2L1 antibody, H2L2 antibody, H2L3 antibody, H2LA1 antibody, H2LA2 antibody, H3L1 antibody, H3L2 antibody, H3LA1 antibody, or H3LA2 antibody, respectively, shown in Tables 3A to 3B.(Appendix 25) The composition according to Appendices 22 or 23, wherein the antibody is an IgM antibody. (Appendix 26) The composition according to any of Appendices 17 to 24, wherein the antibody is a monoclonal antibody. (Appendix 27) The composition according to any of Appendices 17 to 25, wherein the antibody is a humanized antibody. (Appendix 28) The composition according to any of Appendices 17 to 26, wherein the intracranial space is the brain. <Compositions for tissue delivery> (Appendix 29) A composition for use in intraosseous or peripheral nerve delivery, comprising an anti-CADM1 antibody or its antigen-binding fragment and a drug. (Appendix 30) The composition according to Appendices 28, wherein the anti-CADM1 antibody or its antigen-binding fragment binds to an epitope comprising amino acid residues 311, 325, and 326 in CADM1. (Appendix 31) The composition of Appendices 29, wherein the anti-CADM1 antibody or antigen-binding fragment thereof binds to an epitope comprising amino acid residues at positions 311, 312, 325, and 326 in CADM1. (Supplementary Note 32) The anti-CADM1 antibody or antigen-binding fragment thereof has heavy chain CDR1 to 3 and light chain CDR1 to 3, each of which has the amino acid sequence of heavy chain CDR1 to 3 and light chain CDR1 to 3 of 3E1C antibody, 3E1A antibody, 3E1D antibody, 3E1Y antibody, 3E1V antibody, 3E1E antibody, 3E1F antibody, 3E1G antibody, 3E1H antibody, 3E1I antibody, 3E1K antibody, 3E1L antibody, 3E1M antibody, 3E1N antibody, 3E1P antibody, 3E1Q antibody, 3E1R antibody, 3E1S antibody, 3E1W antibody, 3E1T antibody, h3E1A antibody, h3E1F antibody, or h3E1V antibody shown in Table 1A to B; or The composition according to any of Appendices 28 to 30, comprising the amino acid sequences of heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3 of the 45TG2 antibody, 45TG3 antibody, 45TG23 antibody, 45TG24 antibody, 45XL7 antibody, 45XL9 antibody, 45XL11 antibody, 45XL14 antibody, 45XL34 antibody, 45XL45 antibody, 43TG47 antibody, 43XL1 antibody, 43XL4 antibody, 43XL17 antibody, 1-1 antibody, 1-2 antibody, 1-3 antibody, 1-4 antibody, 2-1 antibody, 2-2 antibody, 2-3 antibody, 2-4 antibody, 3-1 antibody, 3-2 antibody, 3-3 antibody, 3-4 antibody, 4-1 antibody, 4-2 antibody, 4-3 antibody, or 4-4 antibody shown in Table 2A to C.(Supplementary Note 33) The anti-CADM1 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region and the light chain variable region have the heavy chain and light chain amino acid sequences of the 3E1C antibody, 3E1A antibody, 3E1D antibody, 3E1Y antibody, 3E1V antibody, 3E1E antibody, 3E1F antibody, 3E1G antibody, 3E1H antibody, 3E1I antibody, 3E1K antibody, 3E1L antibody, 3E1M antibody, 3E1N antibody, 3E1P antibody, 3E1Q antibody, 3E1R antibody, 3E1S antibody, 3E1W antibody, 3E1T antibody, h3E1A antibody, h3E1F antibody, or h3E1V antibody shown in Table 1A to B; or The composition according to any of Appendices 28 to 31, comprising the amino acid sequences of the heavy and light chains of the 45TG2 antibody, 45TG3 antibody, 45TG23 antibody, 45TG24 antibody, 45XL7 antibody, 45XL9 antibody, 45XL11 antibody, 45XL14 antibody, 45XL34 antibody, 45XL45 antibody, 43TG47 antibody, 43XL1 antibody, 43XL4 antibody, 43XL17 antibody, 1-1 antibody, 1-2 antibody, 1-3 antibody, 1-4 antibody, 2-1 antibody, 2-2 antibody, 2-3 antibody, 2-4 antibody, 3-1 antibody, 3-2 antibody, 3-3 antibody, 3-4 antibody, 4-1 antibody, 4-2 antibody, 4-3 antibody, or 4-4 antibody shown in Table 2A to C. (Appendix 34) The composition according to Appendix 28, wherein the anti-CADM1 antibody or antigen-binding fragment thereof has heavy chain CDR1 to 3 and light chain CDR1 to 3 comprising the amino acid sequences of heavy chain CDR1 to 3 and light chain CDR1 to 3 of 9D2 antibody, H1L1 antibody, H1L2 antibody, H1L3 antibody, H1LA1 antibody, H1LA2 antibody, H2L1 antibody, H2L2 antibody, H2L3 antibody, H2LA1 antibody, H2LA2 antibody, H3L1 antibody, H3L2 antibody, H3LA1 antibody, or H3LA2 antibody, respectively, as shown in Tables 3A and 3B. (Appendix 35) The composition according to Appendix 28 or 33, wherein the anti-CADM1 antibody or antigen-binding fragment thereof has a heavy chain variable region and a light chain variable region comprising the amino acid sequences of heavy chain CDR1 to 3 and light chain CDR1 to 3 of 9D2 antibody, H1L1 antibody, H1L2 antibody, H1L3 antibody, H1LA1 antibody, H1LA2 antibody, H2L1 antibody, H2L2 antibody, H2L3 antibody, H2LA1 antibody, H2LA2 antibody, H3L1 antibody, H3L2 antibody, H3LA1 antibody, or H3LA2 antibody, respectively, shown in Tables 3A to 3B.(Appendix 36) The composition according to Appendix 33 or 34, wherein the antibody is an IgM antibody. (Appendix 37) The composition according to any of Appendixes 28 to 35, wherein the antibody is a monoclonal antibody. (Appendix 38) The composition according to any of Appendixes 28 to 36, wherein the antibody is a humanized antibody. <Compositions for delivery to cells> (Appendix 39) A composition for use in delivery to mast cells or osteoblasts, comprising an anti-CADM1 antibody or its antigen-binding fragment and a drug. (Appendix 40) The composition according to Appendix 38, wherein the anti-CADM1 antibody or its antigen-binding fragment binds to an epitope comprising amino acid residues at positions 311, 325, and 326 in CADM1. (Appendix 41) The composition according to Appendix 39, wherein the anti-CADM1 antibody or its antigen-binding fragment binds to an epitope comprising amino acid residues at positions 311, 312, 325, and 326 in CADM1. (Supplementary Note 42) The anti-CADM1 antibody or antigen-binding fragment thereof has heavy chain CDR1 to 3 and light chain CDR1 to 3, each of which has the amino acid sequence of heavy chain CDR1 to 3 and light chain CDR1 to 3 of 3E1C antibody, 3E1A antibody, 3E1D antibody, 3E1Y antibody, 3E1V antibody, 3E1E antibody, 3E1F antibody, 3E1G antibody, 3E1H antibody, 3E1I antibody, 3E1K antibody, 3E1L antibody, 3E1M antibody, 3E1N antibody, 3E1P antibody, 3E1Q antibody, 3E1R antibody, 3E1S antibody, 3E1W antibody, 3E1T antibody, h3E1A antibody, h3E1F antibody, or h3E1V antibody shown in Table 1A to B; or The composition according to any of Appendices 38 to 40, comprising the amino acid sequences of heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3 of the 45TG2 antibody, 45TG3 antibody, 45TG23 antibody, 45TG24 antibody, 45XL7 antibody, 45XL9 antibody, 45XL11 antibody, 45XL14 antibody, 45XL34 antibody, 45XL45 antibody, 43TG47 antibody, 43XL1 antibody, 43XL4 antibody, 43XL17 antibody, 1-1 antibody, 1-2 antibody, 1-3 antibody, 1-4 antibody, 2-1 antibody, 2-2 antibody, 2-3 antibody, 2-4 antibody, 3-1 antibody, 3-2 antibody, 3-3 antibody, 3-4 antibody, 4-1 antibody, 4-2 antibody, 4-3 antibody, or 4-4 antibody shown in Table 2A to C.(Supplementary Note 43) The anti-CADM1 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region and the light chain variable region have the heavy chain and light chain amino acid sequences of the 3E1C antibody, 3E1A antibody, 3E1D antibody, 3E1Y antibody, 3E1V antibody, 3E1E antibody, 3E1F antibody, 3E1G antibody, 3E1H antibody, 3E1I antibody, 3E1K antibody, 3E1L antibody, 3E1M antibody, 3E1N antibody, 3E1P antibody, 3E1Q antibody, 3E1R antibody, 3E1S antibody, 3E1W antibody, 3E1T antibody, h3E1A antibody, h3E1F antibody, or h3E1V antibody shown in Table 1A to B; or The composition according to any of Appendices 38 to 41, comprising the amino acid sequences of the heavy and light chains of the 45TG2 antibody, 45TG3 antibody, 45TG23 antibody, 45TG24 antibody, 45XL7 antibody, 45XL9 antibody, 45XL11 antibody, 45XL14 antibody, 45XL34 antibody, 45XL45 antibody, 43TG47 antibody, 43XL1 antibody, 43XL4 antibody, 43XL17 antibody, 1-1 antibody, 1-2 antibody, 1-3 antibody, 1-4 antibody, 2-1 antibody, 2-2 antibody, 2-3 antibody, 2-4 antibody, 3-1 antibody, 3-2 antibody, 3-3 antibody, 3-4 antibody, 4-1 antibody, 4-2 antibody, 4-3 antibody, or 4-4 antibody shown in Table 2A to C. (Appendix 44) The composition according to Appendix 38, wherein the anti-CADM1 antibody or antigen-binding fragment thereof has heavy chain CDR1 to 3 and light chain CDR1 to 3 comprising the amino acid sequences of heavy chain CDR1 to 3 and light chain CDR1 to 3 of 9D2 antibody, H1L1 antibody, H1L2 antibody, H1L3 antibody, H1LA1 antibody, H1LA2 antibody, H2L1 antibody, H2L2 antibody, H2L3 antibody, H2LA1 antibody, H2LA2 antibody, H3L1 antibody, H3L2 antibody, H3LA1 antibody, or H3LA2 antibody, respectively, as shown in Tables 3A and 3B. (Appendix 45) The composition according to Appendix 38 or 43, wherein the anti-CADM1 antibody or antigen-binding fragment thereof has a heavy chain variable region and a light chain variable region comprising the amino acid sequences of heavy chain CDR1 to 3 and light chain CDR1 to 3 of 9D2 antibody, H1L1 antibody, H1L2 antibody, H1L3 antibody, H1LA1 antibody, H1LA2 antibody, H2L1 antibody, H2L2 antibody, H2L3 antibody, H2LA1 antibody, H2LA2 antibody, H3L1 antibody, H3L2 antibody, H3LA1 antibody, or H3LA2 antibody, respectively, shown in Tables 3A to 3B.(Appendix 46) The composition according to Appendices 43 or 44, wherein the antibody is an IgM antibody. (Appendix 47) The composition according to any of Appendices 38 to 45, wherein the antibody is a monoclonal antibody. (Appendix 48) The composition according to any of Appendices 38 to 46, wherein the antibody is a humanized antibody. <Treatment Method> (Appendix 49) A method for treating a CADM1-related disease, using the pharmaceutical composition according to any of Appendices 1 to 16. (Appendix 50) The treatment method according to Appendices 18, comprising an administration step of administering the antibody or antigen-binding fragment thereof, the antibody-drug conjugate, and / or the pharmaceutical composition to a subject. (Appendix 51) The treatment method according to Appendices 18 or 19, which is used in vitro or in vivo. <Method for Nasal Intracranial Delivery> (Appendix 52) A method for delivering a drug, comprising a step of nasally administering the composition according to any of Appendices 17 to 27 to a subject, thereby delivering the drug intracranially. <Method of delivery to tissue> (Appendix 53) A method of drug delivery, comprising the step of administering to a subject a composition described in any of Appendices 28 to 37, and delivering the drug intrabone or to a peripheral nerve. <Method of delivery to tissue> (Appendix 54) A method of drug delivery, comprising the step of administering to a subject a composition described in any of Appendices 38 to 47, and delivering the drug to mast cells or osteoblasts. <Use> (Appendix 55) A pharmaceutical composition for use in the method of treating a CADM1-associated disease, described in any of Appendices 1 to 16. (Appendix 56) A composition for use in intracranial delivery by nasal administration, described in any of Appendices 17 to 27, and used in a method of intracranial drug delivery by nasal administration. (Appendix 57) A composition for use in intrabone or peripheral nerve delivery, described in any of Appendices 28 to 37, and used in a method of intrabone or peripheral nerve delivery. (Appendix 58) A composition for use in delivery to mast cells or osteoblasts according to any one of Appendices 38 to 47, for use in a method for delivery to mast cells or osteoblasts.

[0282] As described above, the present disclosure provides a pharmaceutical composition comprising a CADM1 antibody or an antigen-binding fragment thereof, which is extremely useful, for example, in the pharmaceutical field.

Claims

1. A pharmaceutical composition for use in treating a CADM1-associated disease, comprising an antibody against cell adhesion molecule 1 (CADM1) or an antigen-binding fragment thereof.

2. The pharmaceutical composition of claim 1, wherein the anti-CADM1 antibody or antigen-binding fragment thereof binds to an epitope comprising amino acid residues 311, 325, and 326 in CADM1.

3. The pharmaceutical composition of claim 2, wherein the anti-CADM1 antibody or antigen-binding fragment thereof binds to an epitope comprising amino acid residues at positions 311, 312, 325, and 326 in CADM1.

4. The anti-CADM1 antibody or antigen-binding fragment thereof, wherein the heavy chain CDR1 to 3 and the light chain CDR1 to 3 are the amino acid sequences of the heavy chain CDR1 to 3 and the light chain CDR1 to 3 of the 3E1C antibody, 3E1A antibody, 3E1D antibody, 3E1Y antibody, 3E1V antibody, 3E1E antibody, 3E1F antibody, 3E1G antibody, 3E1H antibody, 3E1I antibody, 3E1K antibody, 3E1L antibody, 3E1M antibody, 3E1N antibody, 3E1P antibody, 3E1Q antibody, 3E1R antibody, 3E1S antibody, 3E1W antibody, 3E1T antibody, h3E1A antibody, h3E1F antibody, or h3E1V antibody shown in Table 1A to B below, respectively; or The pharmaceutical composition of any one of claims 1 to 3, comprising the amino acid sequences of heavy chain CDR1 to 3 and light chain CDR1 to 3 of the 45TG2 antibody, 45TG3 antibody, 45TG23 antibody, 45TG24 antibody, 45XL7 antibody, 45XL9 antibody, 45XL11 antibody, 45XL14 antibody, 45XL34 antibody, 45XL45 antibody, 43TG47 antibody, 43XL1 antibody, 43XL4 antibody, 43XL17 antibody, 1-1 antibody, 1-2 antibody, 1-3 antibody, 1-4 antibody, 2-1 antibody, 2-2 antibody, 2-3 antibody, 2-4 antibody, 3-1 antibody, 3-2 antibody, 3-3 antibody, 3-4 antibody, 4-1 antibody, 4-2 antibody, 4-3 antibody, or 4-4 antibody, as shown in Tables 2A to 2C below.

5. The anti-CADM1 antibody or antigen-binding fragment thereof, wherein the heavy chain variable region and light chain variable region have the heavy chain and light chain amino acid sequences of 3E1C antibody, 3E1A antibody, 3E1D antibody, 3E1Y antibody, 3E1V antibody, 3E1E antibody, 3E1F antibody, 3E1G antibody, 3E1H antibody, 3E1I antibody, 3E1K antibody, 3E1L antibody, 3E1M antibody, 3E1N antibody, 3E1P antibody, 3E1Q antibody, 3E1R antibody, 3E1S antibody, 3E1W antibody, 3E1T antibody, h3E1A antibody, h3E1F antibody, or h3E1V antibody, respectively, shown in Table 1A to B; or The pharmaceutical composition of any one of claims 1 to 4, comprising the amino acid sequences of the heavy and light chains of 45TG2 antibody, 45TG3 antibody, 45TG23 antibody, 45TG24 antibody, 45XL7 antibody, 45XL9 antibody, 45XL11 antibody, 45XL14 antibody, 45XL34 antibody, 45XL45 antibody, 43TG47 antibody, 43XL1 antibody, 43XL4 antibody, 43XL17 antibody, 1-1 antibody, 1-2 antibody, 1-3 antibody, 1-4 antibody, 2-1 antibody, 2-2 antibody, 2-3 antibody, 2-4 antibody, 3-1 antibody, 3-2 antibody, 3-3 antibody, 3-4 antibody, 4-1 antibody, 4-2 antibody, 4-3 antibody, or 4-4 antibody shown in Tables 2A to 2C.

6. The pharmaceutical composition of claim 1, wherein the anti-CADM1 antibody or antigen-binding fragment thereof comprises heavy chain CDR1-3 and light chain CDR1-3 comprising the amino acid sequences of heavy chain CDR1-3 and light chain CDR1-3 of 9D2 antibody, H1L1 antibody, H1L2 antibody, H1L3 antibody, H1LA1 antibody, H1LA2 antibody, H2L1 antibody, H2L2 antibody, H2L3 antibody, H2LA1 antibody, H2LA2 antibody, H3L1 antibody, H3L2 antibody, H3LA1 antibody, or H3LA2 antibody, respectively, as shown in Tables 3A and 3B below.

7. The pharmaceutical composition of claim 1 or 6, wherein the heavy chain variable region and light chain variable region of the anti-CADM1 antibody or antigen-binding fragment thereof comprise the amino acid sequences of heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3 of 9D2 antibody, H1L1 antibody, H1L2 antibody, H1L3 antibody, H1LA1 antibody, H1LA2 antibody, H2L1 antibody, H2L2 antibody, H2L3 antibody, H2LA1 antibody, H2LA2 antibody, H3L1 antibody, H3L2 antibody, H3LA1 antibody, or H3LA2 antibody, respectively, as shown in Tables 3A to 3B.

8. The pharmaceutical composition according to claim 6 or 7, wherein the antibody is an IgM antibody.

9. A pharmaceutical composition according to any one of claims 1 to 8, which is a monoclonal antibody.

10. A pharmaceutical composition according to any one of claims 1 to 9, which is a humanized antibody.

11. A pharmaceutical composition according to any one of claims 1 to 10, comprising an antibody-drug conjugate comprising an antibody or antigen-binding fragment thereof against CADM1 and a drug.

12. The pharmaceutical composition of any one of claims 1 to 11, wherein the CADM1-related disease is selected from the group consisting of allergic diseases, neurological diseases, bone diseases, type I diabetes, peripheral neuralgia, and pruritus.

13. The pharmaceutical composition according to claim 12, wherein the allergic disease is atopic dermatitis.

14. The pharmaceutical composition of claim 12, wherein the neurological disorder is selected from the group consisting of Alzheimer's disease, stroke, epilepsy, Parkinson's disease, headache, and demyelinating neurological disorders.

15. The pharmaceutical composition of claim 12, wherein the bone disease is selected from the group consisting of osteoporosis, non-healing fractures, and intraosseous non-unions.

16. The pharmaceutical composition of claim 12, wherein the peripheral neuralgia is selected from the group consisting of traumatic pain, chemical traumatic pain, and inflammatory pain.

17. A composition for intracranial delivery via nasal administration, comprising an anti-CADM1 antibody or antigen-binding fragment thereof and a drug.

18. The composition of claim 17, wherein the intracranial space is the brain.

19. A composition for use in intraosseous or peripheral nerve delivery, comprising an anti-CADM1 antibody or an antigen-binding fragment thereof and a drug.

20. A composition for use in delivery to mast cells or osteoblasts, comprising an anti-CADM1 antibody or an antigen-binding fragment thereof and a drug.

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