Mouse FAM167a epitope and monoclonal antibody specifically binding thereto or antigen-binding fragment thereof

Abstract

The present invention relates to a mouse FAM167A epitope and a monoclonal antibody or antigen-binding fragment thereof specifically binding thereto, and more specifically, to a mouse FAM167A epitope comprising the amino acid sequence of SEQ ID NO: 1, a monoclonal antibody or antigen-binding fragment thereof capable of binding specifically to and detecting the epitope, a nucleic acid molecule encoding the heavy chain and / or light chain of the monoclonal antibody or antigen-binding fragment thereof, a vector or host cell comprising the nucleic acid molecule, a method for preparing the monoclonal antibody or antigen-binding fragment thereof, a kit for detecting FAM167A comprising the monoclonal antibody or antigen-binding fragment thereof, and a method for detecting FAM167A using the monoclonal antibody or antigen-binding fragment thereof.

Description

Mouse FAM167A epitope and a monoclonal antibody or antigen-binding fragment thereof that specifically binds thereto

[0001] This application claims priority to Republic of Korea Patent Application No. 10-2024-0093984, filed July 16, 2024, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a mouse FAM167A epitope and a monoclonal antibody or antigen-binding fragment thereof that specifically binds thereto, and more particularly, to a mouse FAM167A epitope comprising an amino acid sequence of SEQ ID NO: 18 and a monoclonal antibody or antigen-binding fragment thereof that specifically binds to and can detect the epitope, a nucleic acid molecule encoding a heavy chain and / or a light chain of the monoclonal antibody or antigen-binding fragment thereof, a vector comprising the nucleic acid molecule, a host cell comprising the nucleic acid molecule, a method for producing the monoclonal antibody or antigen-binding fragment thereof, a kit for detecting FAM167A comprising the monoclonal antibody or antigen-binding fragment thereof, and a method for detecting FAM167A using the monoclonal antibody or antigen-binding fragment thereof.

[0003] The human FAM167A (Family with sequence similarity 167, member A) protein is encoded by the FAM167A gene located on chromosome 8 and is ubiquitously expressed at low levels in all tissue types throughout the body. In mice, it is expressed more highly in the skin, B cells, and spleen, but is equally low in all other cell types.

[0004] According to Non-Patent Documents 1 and 2, a SNP in the region between the FAM167A and BLK genes has been reported to be associated with the development of Sjögren's syndrome not only in the Han Chinese population but also in the Scandinavian population. Furthermore, according to Non-Patent Document 3, as a result of a previous study by the present inventors, it was revealed that FAM167A can be used as an indicator of BCR-ABL-independent tyrosine kinase inhibitor (TKI) resistance in CML that exhibits TKI resistance without mutations in the BCR-ABL kinase domain.

[0005] Although research results on the role of FAM167A in various disease models have been reported, no antibody has yet been developed to detect FAM167A in mice.

[0006] Against this backdrop, the inventors of the present invention have produced a monoclonal antibody that specifically binds to mouse FAM167A to utilize it as a research resource for FAM167A in a disease model using mice, and have confirmed that the produced monoclonal antibody is suitable as an antibody for flow cytometry, western blot, and ELISA (Enzyme Linked Immunosorbent Assay) analysis in a mouse model, thereby completing the present invention.

[0007] [Prior Art Literature]

[0008] [Non-patent literature]

[0009] (Non-patent Document 1) Clinical and Experimental Rheumatology. 31 (5): 704-10.

[0010] (Non-patent literature 2) Genes and Immunity. 12 (2): 100-9

[0011] (Non-patent document 3) Journal of Experimental & Clinical Cancer Research, 41(1), 82.

[0012] The purpose of the present invention is to provide a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to mouse FAM167A.

[0013] Another object of the present invention is to provide a nucleic acid molecule encoding a heavy chain and / or a light chain of the monoclonal antibody or an antigen-binding fragment thereof.

[0014] Another object of the present invention is to provide a vector comprising the nucleic acid molecule and a host cell comprising the vector.

[0015] Another object of the present invention is to provide a method for producing a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to mouse FAM167A using the host cell.

[0016] Another object of the present invention is to provide a kit for detecting FAM167A comprising the aforementioned monoclonal antibody or an antigen-binding fragment thereof.

[0017] Another object of the present invention is to provide a method for detecting FAM167A using the aforementioned monoclonal antibody or antigen-binding fragment thereof.

[0018] Another object of the present invention is to provide an epitope of the mouse FAM167A antigen and a nucleic acid molecule encoding the same, to which the above-described monoclonal antibody or antigen-binding fragment thereof specifically binds.

[0019] In order to solve the above-described problem, the present invention provides a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to mouse FAM167A, comprising a light chain variable region comprising a light chain CDR1 consisting of an amino acid sequence of SEQ ID NO: 1, a light chain CDR2 consisting of an amino acid sequence of SEQ ID NO: 2, and a light chain CDR3 consisting of an amino acid sequence of SEQ ID NO: 3, and a heavy chain variable region comprising a heavy chain CDR1 consisting of an amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 consisting of an amino acid sequence of SEQ ID NO: 5, and a heavy chain CDR3 consisting of an amino acid sequence of SEQ ID NO: 6.

[0020] In the present invention, the monoclonal antibody may include a light chain variable region consisting of an amino acid sequence of SEQ ID NO: 7 and a heavy chain variable region consisting of an amino acid sequence of SEQ ID NO: 8.

[0021] In the present invention, the monoclonal antibody or antigen-binding fragment thereof may be a mouse antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0022] In the present invention, the monoclonal antibody or antigen-binding fragment thereof may be selected from the group consisting of whole IgG, Fab, Fab', F(ab')2, xFab, scFab, dsFv, Fv, scFv-Fc, scFab-Fc, diabody, minibody, scAb, dAb, half-IgG, and combinations thereof.

[0023] In the present invention, the monoclonal antibody or antigen-binding fragment thereof may have a linker consisting of an amino acid sequence of SEQ ID NO: 9 connected between the light chain variable region and the heavy chain variable region.

[0024] In the present invention, the monoclonal antibody or antigen-binding fragment thereof may additionally include a heavy chain constant CH1 fragment consisting of the amino acid sequence of SEQ ID NO: 10.

[0025] In the present invention, the monoclonal antibody or antigen-binding fragment thereof may be a minibody consisting of the amino acid sequence of SEQ ID NO: 11.

[0026] In the present invention, the monoclonal antibody or antigen-binding fragment thereof can be used for flow cytometry analysis, Western blot analysis, or ELISA analysis.

[0027] In the present invention, the monoclonal antibody or antigen-binding fragment thereof can bind to an epitope consisting of the amino acid sequence of SEQ ID NO: 18.

[0028] The present invention also provides a nucleic acid molecule encoding the monoclonal antibody or antigen-binding fragment thereof as described above.

[0029] In the present invention, the nucleic acid molecule encoding the light chain of the monoclonal antibody or antigen-binding fragment thereof may be composed of the nucleotide sequence of SEQ ID NO: 12, and the nucleic acid molecule encoding the heavy chain of the monoclonal antibody or antigen-binding fragment thereof may be composed of the nucleotide sequence of SEQ ID NO: 13.

[0030] In the present invention, when the monoclonal antibody or antigen-binding fragment thereof is a minibody, the nucleic acid molecule encoding the minibody may include the nucleotide sequence of SEQ ID NO: 16.

[0031] Additionally, the present invention provides a vector comprising the above-described nucleic acid molecule and a host cell comprising the same.

[0032] Furthermore, the present invention provides a method for producing a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to mouse FAM167A, which comprises a step of culturing the host cell.

[0033] In addition, the present invention provides a kit for detecting FAM167A comprising the aforementioned monoclonal antibody or an antigen-binding fragment thereof.

[0034] Additionally, the present invention provides the use of the aforementioned monoclonal antibody or antigen-binding fragment thereof for the manufacture of a kit for detecting FAM167A.

[0035] In the present invention, the kit may be an ELISA (Enzyme Linked Immunosorbent Assay) kit.

[0036] In addition, the present invention provides a method for detecting FAM167A protein in a sample or an animal model other than a human, comprising a step of detecting a FAM167A antigen-antibody complex using the aforementioned monoclonal antibody or antigen-binding fragment thereof.

[0037] Additionally, the present invention provides an epitope of a mouse FAM167A antigen comprising an amino acid of SEQ ID NO: 18, to which the aforementioned monoclonal antibody or antigen-binding fragment thereof specifically binds, and a nucleic acid molecule encoding the same.

[0038] Furthermore, the present invention provides the use of an epitope of a mouse FAM167A antigen comprising an amino acid sequence of SEQ ID NO: 18 for the production of the aforementioned monoclonal antibody or an antigen-binding fragment thereof.

[0039] The monoclonal antibody or antigen-binding fragment thereof according to the present invention can specifically bind to mouse FAM167A and effectively detect it, and is suitable for flow cytometry, Western blot analysis, or ELISA analysis, and thus has high value as a research resource for FAM167A in disease models using mice.

[0040] Figure 1 shows the purification results of a monoclonal antibody (anti-mFam167a-1-BSA 2T1A10) according to the present invention.

[0041] Figure 2 shows the results of detecting exogenously expressed mouse FAM167A using a monoclonal antibody (anti-mFam167a) according to the present invention by western blot.

[0042] Figure 3 shows the results of flow cytometry analysis after staining using anti-Myc tag antibody labeled with AF488 for transfection efficiency.

[0043] Figure 4 shows the results of flow cytometry analysis after staining using unlabeled mFam167a antibody and AF488-labeled anti-human IgG antibody.

[0044] Figure 5 shows the results of flow cytometry analysis after staining using mFam167a antibody labeled with AF488 or AF594.

[0045] Figure 6 shows the results of flow cytometry analysis after staining with mFam167a antibody labeled with AF594 and anti-Myc tag antibody labeled with AF488 to confirm co-localization.

[0046] Hereinafter, the present invention will be described in more detail.

[0047] All technical terms used in this invention, unless otherwise defined, have the same meaning as commonly understood by those skilled in the art. While preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of the present invention.

[0048] The term "monoclonal antibody" herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, with the possible exception of variant antibodies, including those containing naturally occurring mutations or those arising during the production of the monoclonal antibody preparation, which mutations are generally present in minor amounts. Unlike polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.

[0049] The term "monospecific" antibody, as used herein, refers to an antibody having more than one binding site, each of which binds to the same epitope of the same antigen.

[0050] The terms "full-length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to antibodies having a structure substantially similar to that of native antibodies. "Native antibodies" refer to naturally occurring immunoglobulin molecules with a variety of structures. For example, native IgG-class antibodies are heterotetrameric glycoproteins of about 150,000 daltons, consisting of two light chains and two heavy chains disulfide-bonded. From N-terminus to C-terminus, each heavy chain has a variable region (VH), called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3), called heavy chain constant regions. Similarly, from N-terminus to C-terminus, each light chain has a variable region (VL), called the variable light domain or light chain variable domain, followed by a light chain constant domain (CL), called the light chain constant region. The heavy chains of antibodies can be assigned to one of five types, called α (IgA), δ (IgD), ε (IgE), γ (IgG), or μ (IgM), some of which can be further classified into subtypes, such as γ1 (IgG1), γ2 (IgG2), γ3 (IgG3), γ4 (IgG4), α1 (IgA1), and α2 (IgA2). The light chains of antibodies can be assigned to one of two types, called kappa (κ) and lambda (λ), based on the amino acid sequence of their constant domains.

[0051] As described above, the variable region allows the antibody to selectively recognize and specifically bind to an epitope on an antigen. That is, the variable region defines a three-dimensional antigen-binding site by combining the VL domain and VH domain, or a subset of the complementarity determining regions (CDRs) of the antibody. This quaternary antibody structure forms the antigen-binding site that is located at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs on each VH and VL chain (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3). In some cases, for example, when a given immunoglobulin molecule is derived from a camelid species or is engineered based on a camelid immunoglobulin, the complete immunoglobulin molecule may be composed solely of heavy chains, without a light chain. See, for example, Hamers-Casterman et al., Nature 363: 446-448 (1993).

[0052] The terms "CDR-H", "HCDR", and "CDRH" are used interchangeably herein to refer to the VH chain of a CDR (e.g., CDR-H1, HCDR1, and CDRH1 refer to the VH1 of a CDR). The terms "CDR-L", "LCDR", and "CDRL" are used interchangeably herein to refer to the VL chain of a CDR (e.g., CDR-L1, LCDR1, and CDRL1 refer to the VL1 of a CDR).

[0053] In naturally occurring antibodies, six "complementarity determining regions", or "CDRs," present in each antigen-binding domain are short, non-contiguous amino acid sequences that are specifically positioned to form the antigen-binding domain as the antibody adopts a three-dimensional conformation in an aqueous environment. The remaining amino acids in the antigen-binding domain, referred to as the "framework" region, exhibit less intermolecular variability. The framework region primarily adopts a β-sheet conformation, and the CDRs form loops that connect them, and in some cases, form part of the β-sheet structure. Thus, the framework region functions to form a scaffold that positions the CDRs in the correct orientation by interchain, non-covalent interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to epitopes on an immunoreactive antigen. This complementary surface facilitates non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDR and framework regions, respectively, for any given heavy or light chain variable region can be readily identified by those skilled in the art, as these are precisely defined (see www.bioinf.org.uk: Dr. Andrew CR Martin's Group; "Sequences of Proteins of Immunological Interest," Kabat, E., et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. MoI. Biol., 196: 901-917 (1987)).

[0054] Where there are two or more definitions for a term used and / or accepted in the art, the definition of the term as used herein is intended to encompass all such meanings unless explicitly stated to the contrary. As a specific example, the term "complementarity determining region" ("CDR") is used to describe the non-contiguous antigen-binding sites found within the variable regions of both heavy and light chain polypeptides. These specific regions are described in Kabat et al., US Dept. of Health and Human Services, "Sequences of Proteins of Immunological Interest" (1983) and Chothia et al., J. Mol. Biol. 196: 901-917 (1987), the entire contents of which are incorporated herein by reference. The CDR definitions according to Kabat and Chothia include overlapping or subsets of amino acid residues when compared to each other. Nonetheless, the application of the definitions to refer to CDRs of an antibody or variant thereof is intended to be within the scope of the terms defined and used herein. Appropriate amino acid residues comprising the CDRs defined by each of the references cited above are provided in Table 1 below for comparison. The exact number of residues comprising a particular CDR will vary depending on the sequence and size of the CDR. One of skill in the art can routinely determine whether a residue comprises a particular CDR by considering the variable region amino acid sequence of the antibody.

[0055] KabatchotiaCDR-H131-3526-32CDR-H150-6552-58CDR-H195-10295-102CDR-L124-3426-32CDR-L250-5650-52CDR-L389-9791-96

[0056] Kabat et al. also defined a numbering system for variable domain sequences applicable to any antibody. One skilled in the art can unambiguously assign the "Kabat numbering" system to any variable domain sequence without relying on any experimental data beyond the sequence itself. As used herein, "Kabat numbering" refers to the numbering system presented in Kabat et al., US Dept. of Health and Human Services, "Sequence of Proteins of Immunological Interest" (1983).

[0057] The antibodies disclosed herein may be derived from any animal source, including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies.

[0058] As used herein, the term "heavy chain constant region" encompasses an amino acid sequence derived from an immunoglobulin heavy chain. As described above, those skilled in the art will appreciate that the heavy chain constant region can be modified to vary its amino acid sequence from that of a naturally occurring immunoglobulin molecule.

[0059] The heavy chain constant region of the antibodies disclosed herein may be derived from different immunoglobulin molecules. For example, the heavy chain constant region of the polypeptide may comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the heavy chain constant region may comprise a hinge region derived in part from an IgG1 molecule and in part from an IgG3 molecule. In another example, the heavy chain portion may comprise a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG4 molecule.

[0060] As used herein, the term "light chain constant region" comprises an amino acid sequence derived from an antibody light chain. Preferably, the light chain constant region comprises at least one of a constant kappa domain or a constant lambda domain.

[0061] A "light chain-heavy chain pair" refers to a collection of light and heavy chains that can form a dimer through a disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0062] "Antibody fragment" or "antigen-binding fragment" refers to a molecule other than an intact antibody, which comprises a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Immunologically functional immunoglobulin fragments include, but are not limited to, Fab, Fab', F(ab')2, xFab, scFab, dsFv, Fv, scFv-Fc, scFab-Fc, diabodies, minibodies, scAbs, dAbs, half-IgGs or combinations thereof. The term "Fab" as used in Fab, Fab', F(ab')2, xFab and scFabs can include conventional Fab fragments and chimeric Fab-like domains as described in PCT / CN2018 / 106766 (Wuxibody). Additionally, it can be derived from any mammal, including but not limited to a human, mouse, rat, camelid, or rabbit. A functional portion of an antibody, such as one or more of the CDRs described herein, can be covalently linked to a second protein or small molecule compound, thereby enabling it to be used as a targeted therapeutic for a specific target. The term "antibody fragment" includes aptamers, spiegelmers, and diabodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen and forming a complex.

[0063] Antibody fragments can be prepared by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies as described herein, as well as production in recombinant host cells, such as E. coli or phage.

[0064] Papain digestion of intact antibodies produces two identical antigen-binding fragments, called "Fab" fragments, which contain the heavy and light chain variable domains and the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Therefore, as used herein, the term "Fab fragment" refers to a light chain fragment comprising the VL domain and the constant domain (CL) of the light chain, and an antibody fragment comprising the VH domain and the first constant domain (CH1) of the heavy chain. A Fab' fragment differs from a Fab fragment in that it adds several residues to the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is a Fab' fragment in which the cysteine ​​residue(s) of the constant domains have a free thiol group. Pepsin treatment produces an F(ab')2 fragment, which has two antigen-binding sites (two Fab fragments) and part of the Fc region. As used herein, the "F(ab')2 fragment" comprises, as described above, two light chains and two heavy chains comprising a variable region, CH1, and a portion of a constant region between the CH1 and CH2 domains, thereby forming an intrachain disulfide bond between the two heavy chains. Accordingly, the F(ab')2 fragment is composed of two Fab' fragments, and the two Fab' fragments are linked to each other by a disulfide bond therebetween.

[0065] The term "cross-Fab fragment" or "xFab fragment" or "crossover Fab fragment" refers to a Fab fragment in which the variable or constant regions of the heavy and light chains are exchanged. Two different chain compositions of crossover Fab molecules are possible and are included in the bispecific antibodies of the invention: on the one hand, the variable regions of the Fab heavy and light chains are exchanged, i.e. the crossover Fab molecule comprises a peptide chain composed of a light chain variable region (VL) and a heavy chain constant region (CH1), and a peptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL). Such a crossover Fab molecule is also called CrossFab (VLVH). In contrast, when the constant regions of the Fab heavy and light chains are exchanged, the crossover Fab molecule comprises a peptide chain composed of the heavy chain variable region (VH) and the light chain constant region (CL), and a peptide chain composed of the light chain variable region (VL) and the heavy chain constant region (CH1). Such a crossover Fab molecule is also called CrossFab(CLCH1).

[0066] A "single chain Fab fragment" or "scFab" is a polypeptide comprising an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein the antibody domains and the linker have one of the following sequences from N-terminus to C-terminus: a) VH-CH1-linker-VL-CL, b) VL-CL-linker-VH-CH1, c) VH-CL-linker-VL-CH1 or d) VL-CH1-linker-VH-CL; and wherein the linker is a polypeptide having at least 30 amino acids, preferably 32 to 50 amino acids. The single chain Fab fragment is stabilized by a natural disulfide bond between the CL domain and the CH1 domain. Additionally, these single-chain Fab molecules can be further stabilized by the creation of interchain disulfide bonds through insertion of cysteine ​​residues (e.g., at position 44 in the variable heavy chain and position 100 in the variable light chain according to Kabat numbering).

[0067] A "crossover single chain Fab fragment" or "x-scFab" is a polypeptide comprising an antibody heavy chain variable domain (VH), an antibody constant domain 1 (CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein the antibody domains and the linker have one of the following sequences from N-terminus to C-terminus: (a) VH-CL-linker-VLCH1 and (b) VL-CH1-linker-VH-CL; wherein the VH and VL together form an antigen-binding domain that specifically binds to an antigen, and wherein the linker is a polypeptide having at least 30 amino acids. Additionally, these x-scFab molecules can be further stabilized by formation of an interchain disulfide bond through insertion of a cysteine ​​residue (e.g., at position 44 in the variable heavy chain and position 100 in the variable light chain according to the Kabat numbering).

[0068] An "Fv region" is an antibody that includes the variable regions of each heavy and light chain, but not the constant region. An scFv is an Fv linked by a flexible linker. An scFv-Fc is an Fc linked to an scFv. A minibody typically has a CH3 fragment linked to an scFv, but a minibody according to the present invention may have a CH1 fragment linked to an scFv. A diabody includes two molecules of an scFv. A "single-chain variable fragment" or "scFv" refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of an immunoglobulin. In some embodiments, the regions are linked to a short linker peptide having from about 10 to about 25 amino acids. The linker can be glycine-rich for flexibility, serine-rich for solubility, or threonine-rich for solubility, and can link the N-terminus of the VH to the C-terminus of the VL, or vice versa. These proteins retain the specificity of the native immunoglobulin despite the removal of the constant region and the introduction of the linker. ScFv molecules are known in the art and are described, for example, in U.S. Patent No. 5,892,019.

[0069] A "short-chain antibody (scAb)" is a single polypeptide chain comprising one heavy chain variable region or one light chain constant region, with the heavy and light chain variable regions connected by a flexible linker. See, for example, U.S. Patent No. 5,260,203, which is incorporated herein by reference.

[0070] A "domain antibody (dAb)" is an immunologically functional immunoglobulin fragment comprising only the variable region of a heavy chain or the variable region of a light chain. In one embodiment, two or more VH regions are covalently linked by a peptide linker to form a bivalent domain antibody. The two VH regions of such a bivalent domain antibody may target the same or different antigens.

[0071] The term "full-length IgG" according to the present invention is defined as comprising essentially complete IgG, but does not necessarily have all the functions of a complete IgG. For the avoidance of doubt, a full-length IgG contains two heavy chains and two light chains. Each chain contains constant (C) and variable (V) regions, which can be divided into domains designated CH1, CH2, CH3, VH, and CL, VL. IgG antibodies bind to antigens through the variable region domains contained in the Fab portion, and after binding, can interact with cells and molecules of the immune system through the constant domains, mostly through the Fc portion. The terms 'variable region domain', 'variable region', 'variable domain', 'VH / VL pair', 'VH / VL', 'Fab portion', 'Fab arm', 'Fab' or 'arm' are used interchangeably herein. A full-length antibody according to the present invention includes an IgG molecule that may have mutations that provide the desired characteristics. Such mutations must not result in the deletion of a significant portion of any region. However, an IgG molecule in which one or more amino acid residues are deleted without substantially altering the binding properties of the resulting IgG molecule is included within the term "full-length IgG". For example, such an IgG molecule may have one or more deletions of 1 to 10 amino acid residues, preferably in a non-CDR region, wherein the deletion of the amino acid is not essential for the binding specificity of the IgG.

[0072] As used herein, the term "antigen binding domain" or "antigen-binding site" refers to the portion of an antibody or antibody fragment that specifically binds to an antigenic determinant. More specifically, the term "antigen binding domain" refers to the portion of an antibody that specifically binds to and is complementary to part or all of an antigen. When the antigen is large, the antibody or antibody fragment may bind only to a specific portion of the antigen, which portion is called an epitope. The antigen binding domain may be provided, for example, by one or more variable domains (also referred to as variable regions). Preferably, the antigen binding domain comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). In one aspect, the antigen binding domain can bind to its antigen and block or partially block its function. Antigen binding domains that specifically bind FAM167A include antibodies and fragments thereof as further defined herein. Additionally, the antigen binding domain may comprise a scaffold antigen binding protein, e.g., a binding domain based on a designed repeat protein or a designed repeat domain (see e.g. WO 2002 / 020565).

[0073] As used herein, the term "antigenic determinant" is synonymous with "antigen" and "epitope" and refers to a site on a polypeptide macromolecule (e.g., a contiguous stretch of amino acids or a conformational configuration comprised of different regions of non-contiguous amino acids) to which an antigen-binding moiety binds to form an antigen-binding moiety-antigen complex. Useful antigenic determinants can be found, for example, on the surface of tumor cells, the surface of virus-infected cells, the surface of other diseased cells, the surface of immune cells, in serum-free blood, and / or in the extracellular matrix (ECM). Unless otherwise specified, a protein useful as an antigen herein can be any native form of the protein from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats). In a particular embodiment, the antigen is a human or mouse protein. When referring to a specific protein herein, the term encompasses both the "full-length," unprocessed protein, as well as all forms of the protein produced by processing in cells. The term also encompasses naturally occurring variants of the protein, such as splice variants or allelic variants.

[0074] "Specific binding" means that binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antibody or antibody fragment to bind a specific antigen can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) technology (analyzed on a BIAcore instrument) (Liljeblad et al., Glyco J 17, 323-329 (2000)), and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)).

[0075] "Affinity" or "binding affinity" refers to the strength of the sum of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). As used herein, unless otherwise specified, "binding affinity" refers to the intrinsic binding affinity reflecting a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed in terms of the dissociation constant (Kd), which is the ratio of the dissociation rate constant and the association rate constant (koff and kon, respectively). Thus, equivalent affinities can include different rate constants, as long as the ratio of the rate constants remains the same. Affinity can be measured by routine methods known in the art, including those described herein. A particular method for measuring affinity is surface plasmon resonance (SPR).

[0076] The terms “anti-FAM167A antibody,” “antibody that specifically binds to FAM167A,” and “antibody comprising an antigen-binding domain that specifically binds to FAM167A” refer to antibodies that can bind to FAM167A with sufficient affinity to enable detection of FAM167A, such as by Western blot, ELISA, and flow cytometry.

[0077] The term "mouse" antibody is intended to encompass antibodies having variable regions in which both the framework and CDR regions are derived from mouse germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from mouse germline immunoglobulin sequences. The mouse antibodies of the present disclosure may comprise amino acid residues not encoded by mouse germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by in vivo complementary somatic mutagenesis).

[0078] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, and the remainder of the heavy and / or light chain is derived from a different source or species.

[0079] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains corresponding to the various classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0080] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody.

[0081] A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant region has been further modified or altered from the constant region of the original antibody to produce the properties of the present invention, particularly with respect to C1q binding and / or Fc receptor (FcR) binding.

[0082] A "human" antibody is one that has an amino acid sequence corresponding to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire or other human antibody-coding sequences. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues.

[0083] The term "Fc domain" or "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions.

[0084] The term "linker" refers to a peptide comprising one or more amino acids, typically about 2 to 20 amino acids. Any peptide linker known in the art can be used as the linker. The peptide linker separates the light chain variable domain and the heavy chain variable domain by a sufficient distance to allow each variable domain to fold into appropriate secondary and tertiary structures. The sequence of a suitable peptide linker can be selected by considering the following factors: (a) the ability to have a flexible extended conformation; (b) the ability to not create secondary structures that interact with the epitope; and (c) the absence of hydrophobic residues or charged residues that can react with the epitope. Preferred peptide linkers include Gly, Glu, Asn, Lys, Ser, and Pro residues. Other neutral amino acids, such as Thr and Ala, can also be included in the linker sequence. The linker sequence may consist of 1-50 amino acid residues, preferably 10-20 amino acid residues. For example, a suitable non-immunogenic linker peptide is, for example, (G4S). n , (SG4) n or G4(SG4) n The amino acid sequence may include, but is not limited to, the amino acid sequence of SEQ ID NO: 9. Here, "n" may generally be an integer from 1 to 10, typically from 2 to 4. A preferred linker sequence according to one embodiment of the present invention may include or consist of the amino acid sequence of SEQ ID NO: 9.

[0085] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage by which the amino acid residues in the candidate sequence are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without considering conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways within the art, for example, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software, or the FASTA program package. One skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithm necessary to achieve maximum alignment over the entire length of the compared sequences. However, for purposes herein, percent amino acid sequence identity values ​​are generated using the ggsearch program in the FASTA package, version 36.3.8c or later, in conjunction with a BLOSUM50 comparison matrix. The FASTA program package is described in WR Pearson and DJ Lipman (1988), "Improved Tools for Biological Sequence Analysis," PNAS 85:2444-2448; WR Pearson (1996), "Effective Protein Sequence Comparison," Meth. Enzymol. 266:227-258; and Pearson et al. (1997) Genomics 46:24-36, and is publicly available at http: / fasta.bioch.virginia.edu / fasta_www2 / fasta_down.shtml. Alternatively, see http: / fasta.bioch.virginia.edu / fasta_www2 / index.You can compare sequences using a public server accessible from cgi, and use the ggsearch(global protein:protein) program with default options (BLOSUM50; open: -10; ext: -2; Ktup=2) to perform a global alignment rather than a local one. The percent amino acid identity (%) is provided in the output alignment header.

[0086] As used herein, the term "polypeptide" is intended to encompass not only a singular "polypeptide" but also a plural "polypeptides," and refers to a molecule comprising monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids are encompassed within the definition of "polypeptide," and the term "polypeptide" may be used in place of, or interchangeably with, any of these terms. The term "polypeptide" is also intended to refer to the product of post-expression modification of a polypeptide, including but not limited to glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. Polypeptides may be derived from natural biological sources or produced by recombinant techniques, but are not necessarily translated from a designated nucleic acid sequence. They may be produced by any means, including chemical synthesis. The term "polypeptide" also encompasses variants and derivatives of polypeptides. Furthermore, "polypeptide fragment" refers to a polypeptide having a deletion of the amino-terminal amino acid sequence, a deletion of the carboxyl-terminal amino acid sequence, and / or an internal deletion, compared to the full-length protein. Such fragments may also contain modified amino acids compared to the full-length protein.In one embodiment, the fragment can be about 5 to 900 amino acids in length, for example at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 or more amino acids in length. For the purposes of the present invention, useful polypeptide fragments include immunologically functional fragments of antibodies comprising an antigen-binding domain. For FAM167A binding antibodies, such useful fragments include, but are not limited to, all or part of an antibody chain comprising one, two or three heavy or light chain CDR sequences, or a variable or constant region of a heavy or light chain.

[0087] As used herein, a "variant" of a polypeptide, such as, for example, an antigen-binding fragment, protein, or antibody, is a polypeptide having one or more amino acid residues inserted, deleted, added, and / or substituted as compared to another polypeptide sequence, including a fusion polypeptide. Protein variants also include those that have been modified by proteolytic enzyme cleavage, phosphorylation, or other post-translational modification, yet retain the biological activity of an antibody disclosed herein, such as specific binding to FAM167A and biological activity. A variant can be about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% identical to the sequence of an antibody or antigen-binding fragment thereof disclosed herein.

[0088] As used herein, the term "recombinant" with respect to a polypeptide or polynucleotide means a form of a polypeptide or polynucleotide that does not exist in nature, a non-limiting example of which can be formed by combining polynucleotides or polypeptides that do not normally exist together.

[0089] "Homology" or "identity" or "similarity" refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned for comparison purposes. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of positions shared by the sequences that are identical or homologous. An "unrelated" or "non-homologous" sequence shares less than 40% identity, and preferably less than 25% identity, with one of the sequences of the present disclosure.

[0090] A polynucleotide or polynucleotide region (or polypeptide or polypeptide region) having a given percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" to another sequence means that when two sequences are aligned, the bases (or amino acids) are the same by that percentage.

[0091] The term "polynucleotide" refers to an isolated nucleic acid molecule or structure, such as messenger RNA (mRNA), virally derived RNA, or plasmid DNA (pDNA). A polynucleotide may comprise conventional phosphodiester linkages or non-conventional linkages (e.g., amide linkages, such as those found in peptide nucleic acids (PNA). The term "nucleic acid molecule" refers to any one or more nucleic acid segments, such as DNA or RNA fragments, present in a polynucleotide.

[0092] An "isolated" nucleic acid molecule or polynucleotide refers to a nucleic acid molecule, DNA, or RNA, obtained from its natural environment. For example, a recombinant polynucleotide encoding a polypeptide contained in a vector is considered isolated for the purposes of the present invention. Additional examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or polynucleotides purified (partially or substantially) in solution. An isolated polynucleotide generally includes a polynucleotide molecule contained in a cell containing the polynucleotide molecule, but the polynucleotide molecule is present extrachromosomally or at a chromosomal location different from the natural chromosomal location. Isolated RNA molecules include the in vivo or in vitro RNA transcripts of the present invention, as well as positive and negative strand forms, and double-stranded forms. Isolated polynucleotides or nucleic acids according to the present invention further include synthetically produced molecules. Additionally, the polynucleotide or nucleic acid may be or include regulatory elements such as a promoter, ribosome binding site, or transcription terminator. The term "isolated" as used herein also refers to a nucleic acid or peptide being substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. The term "isolated" is also used herein to refer to a cell or polypeptide being separated from other cellular proteins or tissues. Isolated polypeptides are meant to include both purified polypeptides and recombinant polypeptides.

[0093] The term "expression cassette" refers to a polynucleotide produced recombinantly or synthetically using a series of specified nucleic acid elements that enable transcription of a specific nucleic acid in a target cell. The recombinant expression cassette may be incorporated into a plasmid, chromosome, mitochondrial DNA, plasmid DNA, virus, or nucleic acid fragment. Typically, the recombinant expression cassette portion of an expression vector comprises, among other things, the nucleic acid sequence to be transcribed and a promoter. In certain embodiments, the expression cassette comprises a polynucleotide sequence encoding a monoclonal antibody of the invention or an antigen-binding fragment thereof.

[0094] The term "vector" or "expression vector" refers to a DNA molecule used to introduce and express a specific gene operably linked to a cell. The term encompasses vectors as self-replicating nucleic acid structures as well as vectors introduced by integration into the genome of a host cell. The expression vector of the present invention comprises an expression cassette. The expression vector enables the transcription of large quantities of stable mRNA. When the expression vector is present within a cell, the ribonucleic acid molecule or protein encoded by the gene is produced by the cellular transcription and / or translation machinery. In one embodiment, the expression vector of the present invention comprises an expression cassette comprising a polynucleotide sequence encoding a monoclonal antibody of the present invention or an antigen-binding fragment thereof.

[0095] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced (including the progeny of such cells). Host cells include "transformants" and "transformed cells," which include primary transformed cells and their derived progeny, regardless of the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as that selected or selected from the originally transformed cell are included herein. A host cell is any type of cell system that can be used to produce an antibody or bispecific antibody of the invention. Host cells include cultured cells, for example mammalian cultured cells such as HEK cells, CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, P3X63 mouse myeloma cells, PER cells, PER.C6 cells or hybridoma cells, yeast cells, insect cells, and plant cells, including cells contained within transgenic animals, transgenic plants or cultured plant or animal tissues.

[0096] A first aspect of the present invention relates to a monoclonal antibody or antigen-binding fragment thereof that specifically binds to FAM167A.

[0097] The monoclonal antibody or antigen-binding fragment thereof that specifically binds to FAM167A according to the present invention has the property of binding to the FAM167 protein with high affinity, and in one embodiment, the monoclonal antibody or antigen-binding fragment thereof according to the present invention may include a light chain variable region and a heavy chain variable region defined as follows (a) and (b):

[0098] (a) a light chain variable region comprising a light chain CDR1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 1, a light chain CDR2 (LCDR2) comprising the amino acid sequence of SEQ ID NO: 2, and a light chain CDR3 (LCDR3) comprising the amino acid sequence of SEQ ID NO: 3; and

[0099] (b) A heavy chain variable region comprising a heavy chain CDR1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 (HCDR2) comprising the amino acid sequence of SEQ ID NO: 5, and a heavy chain CDR3 (HCDR3) comprising the amino acid sequence of SEQ ID NO: 6.

[0100] A monoclonal antibody or antigen-binding fragment thereof according to one embodiment of the present invention may include a light chain variable region comprising LCDR1, LCDR2, and LCDR3 each consisting of the amino acid sequences described in (a) above; and a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 each consisting of the amino acid sequences described in (b) above.

[0101] In another embodiment, the monoclonal antibody or antigen-binding fragment thereof may comprise a light chain variable region and a heavy chain variable region defined as follows (c) and (d):

[0102] (c) a light chain variable region (VL) comprising the amino acid sequence of sequence number 7; and

[0103] (d) A heavy chain variable region (VH) comprising the amino acid sequence of sequence number 8.

[0104] According to another embodiment, a monoclonal antibody or antigen-binding fragment thereof may comprise a VL consisting of the amino acid sequence described in (c) above and a VH consisting of the amino acid sequence described in (d) above.

[0105] The above "FAM167A", also known as C8orf13, is a 214 amino acid protein belonging to the FAM167 (SEC) family. The gene encoding FAM167A is mapped to human chromosome 8, which consists of approximately 146 million base pairs. The FAM167A protein is not particularly limited in type, but may preferably be a mouse FAM167A protein. The mouse FAM167A protein or a fragment thereof may include or consist of the amino acid sequence disclosed in GenBank: AAH65085.1 or a portion thereof, but may include, without limitation, an amino acid sequence that exhibits substantially the same or corresponding efficacy as the protein. The FAM167A protein may also include an isoform or a precursor thereof. In the present invention, the amino acid sequence of a representative mouse FAM167A protein is shown in SEQ ID NO: 17.

[0106] MSVPQIQVEE VSEKDRPAGA AVPPDDHLLS LKALTEKLRL ETRRPSYLEW QARLEEQTWP FPRPAAQQEA SLEQGACGGG EPLMPLKEPR DLLPPSASAG RGDRPLTTGK LEGFQSIDEA IAWLRKELAE MRLQDQQLAR QLMRLRGDIN KLKIEQTCRL HRRMLNDAAF ELEERDELSD LFCDSPLASS FSLSMPLKLI GVTKMNINSR RFSLC (SEQ ID NO: 17)

[0107] The monoclonal antibody or antigen-binding fragment thereof according to the present invention may be a mouse antibody, a chimeric antibody, a humanized antibody or a fully human antibody.

[0108] In the present invention, the monoclonal antibody or antigen-binding fragment thereof may be selected from the group consisting of whole IgG, Fab, Fab', F(ab')2, xFab, scFab, dsFv, Fv, scFv-Fc, scFab-Fc, diabody, minibody, scAb, dAb, half-IgG, and combinations thereof. Preferably, it may be a minibody in which a fragment of scFv and IgG1-Fc (e.g., CH1) is bound, and may have a sequence of a humanized antibody or a human antibody.

[0109] In one embodiment of the present invention, the form of the minibody may be one in which a CH1 fragment of a light chain variable region, a linker, a heavy chain variable region, and a heavy chain constant region are sequentially connected.

[0110] In one embodiment of the present invention, the linker may be a peptide linker comprising or consisting of the amino acid sequence of SEQ ID NO: 9.

[0111] In one embodiment of the present invention, the CH1 fragment of the heavy chain constant region may comprise or consist of the amino acid sequence of SEQ ID NO: 10.

[0112] According to a preferred embodiment of the present invention, a monoclonal antibody or an antigen-binding fragment thereof may be a minibody comprising or consisting of the amino acid sequence of SEQ ID NO: 11.

[0113] In the present invention, the monoclonal antibody or antigen-binding fragment thereof may include a variant of an amino acid sequence having the characteristics of the VL, VH, linker, and CH1 described above. For example, the amino acid sequence of the antibody may be changed to improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the antibody. Such amino acid mutations are made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; And phenylalanine, tryptophan, and tyrosine are biologically functional equivalents.

[0114] Amino acid exchanges in proteins that do not alter the overall activity of the molecule are well known in the art (H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979). The most common exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, Asp / Gly.

[0115] Considering the mutations having the above-described biological equivalent activity, the monoclonal antibody or antigen-binding fragment thereof of the present invention and the nucleic acid molecule encoding the same are interpreted to also include sequences that exhibit substantial identity with the sequences listed in the sequence listing. The substantial identity refers to a sequence that exhibits at least 60% identity, more preferably 70% identity, even more preferably 80% identity, and most preferably at least 90% identity when the sequence of the present invention and any other sequence are aligned to the greatest extent possible and the aligned sequences are analyzed using an algorithm commonly used in the art.

[0116] In one embodiment, the monoclonal antibody or antigen-binding fragment thereof can comprise a peptide having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence defined above.

[0117] In one embodiment of the present invention, the monoclonal antibody or antigen-binding fragment thereof may be used for flow cytometry, Western blot analysis, or ELISA (Enzyme Linked Immunosorbent Assay) analysis, but is not limited thereto. For example, the monoclonal antibody or antigen-binding fragment thereof of the present invention may be used without limitation in any method for detecting the FAM167A protein in a given sample or an animal model of a specific disease.

[0118] In the present invention, the monoclonal antibody or antigen-binding fragment thereof can bind to an epitope consisting of the amino acid sequence of SEQ ID NO: 18.

[0119] A second aspect of the present invention relates to a nucleic acid molecule encoding a monoclonal antibody or an antigen-binding fragment thereof according to the present invention, a vector comprising the nucleic acid molecule, and a host cell comprising the vector.

[0120] The nucleic acid molecule of the present invention is an isolated nucleic acid molecule. In one embodiment of the present invention, the nucleic acid molecule encoding the light chain of the monoclonal antibody or antigen-binding fragment thereof may comprise or consist of the nucleotide sequence of SEQ ID NO: 12, and the nucleic acid molecule encoding the heavy chain of the monoclonal antibody or antigen-binding fragment thereof may comprise or consist of the nucleotide sequence of SEQ ID NO: 13.

[0121] When the monoclonal antibody or antigen-binding fragment thereof according to a preferred embodiment of the present invention is a minibody, the nucleic acid molecule encoding the minibody may comprise or consist of the nucleotide sequence of SEQ ID NO: 16.

[0122] The nucleic acid molecule of the present invention is also interpreted to include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence described above. The substantial identity refers to a nucleotide sequence that exhibits at least 80% identity, more preferably at least 90% identity, when the nucleotide sequence of the present invention is arranged to correspond as much as possible to any other sequence and the arranged sequence is analyzed using an algorithm commonly used in the art.

[0123] In one embodiment, the nucleic acid molecule can comprise a nucleic acid molecule having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence defined above.

[0124] In the present invention, the vector includes a plasmid vector; a phagemid vector; a cosmid vector; and a viral vector such as a bacteriophage vector, an adenovirus vector, a retrovirus vector, and an adeno-associated virus vector, and preferably a plasmid vector.

[0125] The vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are disclosed in Sambrook et al. (2001), Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, which is incorporated herein by reference.

[0126] The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host.

[0127] When the vector of the present invention is an expression vector and uses a prokaryotic cell as a host, it generally includes a strong promoter capable of initiating transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. When E. coli (e.g., BL21, HB101, DH5α, etc.) is used as a host cell, E. The promoter and operator regions of the tryptophan biosynthetic pathway of B. coli (Yanofsky, C. (1984), J. Bacteriol., 158:1018-1024) and the left-hand promoter of phage λ (pLλ promoter, Herskowitz, I. and Hagen, D. (1980), Ann. Rev. Genet., 14:399-445) can be used as regulatory regions.

[0128] Vectors that can be used in the present invention can be produced by manipulating plasmids (e.g., pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series (pET28a, pET21a, etc.) and pUC19, etc.), phagemids (e.g., pComb3X), phages (e.g., λgt4·λB, λ-Charon, λΔz1 and M13, etc.) or viruses (e.g., SV40, etc.) that are frequently used in the art.

[0129] When the vector of the present invention is an expression vector and uses a eukaryotic cell as a host, a promoter derived from the genome of a mammalian cell (e.g., metallothionine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus promoter, and HSV late promoter) can be used, and generally has a polyadenylation sequence as a transcription termination sequence.

[0130] The vector of the present invention may be fused with other sequences as needed to facilitate purification of the amino-terminal protein of the protein expressed therefrom. The sequences to be fused include, but are not limited to, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA).

[0131] The expression vector of the present invention may include an antibiotic resistance gene commonly used in the art as a selectable marker, for example, a resistance gene for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0132] The vector expressing the monoclonal antibody of the present invention or its antigen-binding fragment can be either a vector system in which the light chain and the heavy chain are simultaneously expressed from a single vector, or a system in which the light chain and the heavy chain are each expressed from separate vectors. In the latter case, the two vectors are introduced into a host cell through co-transformation and targeted transformation. Co-transformation is a method in which each vector DNA encoding the light chain and the heavy chain is simultaneously introduced into a host cell, and then cells expressing both the light chain and the heavy chain are selected. Targeted transformation is a method in which cells transformed with a vector containing a light chain (or heavy chain) are selected, and the selected cells expressing the light chain are transformed again with a vector containing a heavy chain (or light chain), thereby finally selecting cells expressing both the light chain and the heavy chain.

[0133] The host cell according to one embodiment of the present invention is a cell transformed with the above-described vector. Any host cell known in the art that can stably and continuously clone and express the vector of the present invention can be used, and examples thereof include, but are not limited to, prokaryotic host cells such as Bacillus spp. such as Escherichia coli, Bacillus subtilis and B. thuringensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis or Staphylococcus (e.g., Staphylococcus carnosus).

[0134] Suitable eukaryotic host cells for the above vector include multicellular fungi such as Aspergillus spp. and Neurospora crassa belonging to the Phylum Ascomycota, unicellular fungi including yeasts such as Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces, other lower eukaryotic cells, higher eukaryotic cells such as insect-derived cells, and cells derived from plants or mammals.

[0135] In the present invention, the term "transfection" refers to introducing a desired gene into a host cell using the recombinant vector of the present invention, and is used with the same meaning as "transformation." Therefore, "transfection" and / or "transformation" into a host cell includes any method for introducing a nucleic acid into an organism, cell, tissue, or organ, and can be performed by selecting an appropriate standard technique depending on the host cell as is known in the art. Such methods include, but are not limited to, electroporation, protoplast fusion, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, stirring using silicon carbide fibers, Agrobacterium-mediated transformation, PEG, dextran sulfate, lipofectamine, and desiccation / inhibition-mediated transformation methods.

[0136] A third aspect of the present invention relates to a method for producing a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to FAM167A, comprising a step of culturing the aforementioned host cell.

[0137] Host cell culture for the production of antibodies or antigen-binding fragments thereof can be performed using appropriate media and culture conditions known in the art. These culture processes can be easily adjusted and utilized by those skilled in the art depending on the selected strain. Cell culture is categorized into suspension culture and adherent culture based on cell growth mode, and batch, fed-batch, and continuous culture based on culture method. The culture medium used must adequately satisfy the requirements of the specific strain.

[0138] The medium used for culturing animal cells contains various carbon sources, nitrogen sources, and trace element components. Examples of carbon sources that can be used include carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch, and cellulose; fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These carbon sources can be used alone or in combination. Examples of nitrogen sources that can be used include organic nitrogen sources such as peptone, yeast extract, meat juice, malt extract, corn steep liquor (CSL), and soybean meal; and inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and ammonium nitrate. These nitrogen sources can be used alone or in combination. The above-mentioned medium may contain, as personnel, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and the corresponding sodium-containing salt. It may also contain a metal salt such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins, and suitable precursors may be included.

[0139] During cultivation, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid, and sulfuric acid can be appropriately added to the culture to adjust the pH of the culture. Furthermore, foaming can be suppressed during cultivation using antifoaming agents such as fatty acid polyglycol esters. Furthermore, oxygen or an oxygen-containing gas (e.g., air) is injected into the culture to maintain an aerobic state. The culture temperature is usually between 20°C and 45°C, preferably between 25°C and 40°C.

[0140] Antibodies obtained by culturing host cells can be used in an unpurified state, or can be further purified to high purity using various conventional methods, such as dialysis, salt precipitation, and chromatography. Among these, the method using chromatography is the most commonly used, and the type and order of the column can be selected from ion exchange chromatography, size exclusion chromatography, and affinity chromatography, depending on the characteristics of the antibody, the culturing method, etc.

[0141] The fourth aspect of the present invention relates to a kit for detecting FAM167A comprising the above-described monoclonal antibody or an antigen-binding fragment thereof and a method for detecting FAM167A using the above-described monoclonal antibody or an antigen-binding fragment thereof.

[0142] In connection with the fourth aspect, the present invention provides the use of the above-described monoclonal antibody or antigen-binding fragment thereof for the manufacture of a kit for detecting FAM167A.

[0143] In one embodiment of the present invention, the kit may be in the form of an ELISA (Enzyme-linked immunosorbent assay) kit, and specifically, the kit may include the above-described monoclonal antibody or an antigen-binding fragment thereof as a capture antibody, and the ELISA may include, but is not limited to, (i) a polyclonal antibody that binds to FAM167A; (ii) a capture antibody in the form of a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to the FAM167A protein according to the present invention; and (iii) an antibody bound to an IgG Fc to which a detection label is bound.

[0144] The term "ELISA (Enzyme-linked immunosorbent assay)" of the present invention is also called an enzyme-linked immunosorbent assay, and is a method of quantifying by using absorbance through a reaction between an enzyme and a substrate by forming an antigen-antibody complex by binding an enzyme to an antibody. The ELISA includes a direct ELISA using a labeled secondary antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled secondary antibody that recognizes a capture antibody in a complex of antibodies that recognize the antigen attached to the solid support, a direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of antibodies and antigens attached to the solid support, an indirect sandwich ELISA using a labeled secondary antibody that recognizes an antibody after reacting with another antibody that recognizes an antigen in a complex of antibodies and antigens attached to the solid support, and a competitive ELISA that competes between different antigens having the same antibody binding site.

[0145] The kit of the present invention may additionally include tools or reagents known in the art for use in immunological analysis in addition to antibodies against FAM167A.

[0146] Tools or reagents used in immunological analysis may include suitable carriers or supports, labels capable of generating detectable signals, solubilizers, detergents, and stabilizers. Suitable carriers may also include, but are not limited to, a substrate capable of measuring enzyme activity when the label is an enzyme, a suitable buffer solution, a secondary antibody labeled with a chromogenic enzyme or fluorescent substance, a chromogenic substrate, and a reaction stopper.

[0147] The antibody against FAM167A included in the kit of the present invention can preferably be immobilized on a suitable carrier or support using various methods as disclosed in the literature, examples of suitable carriers or supports include PBS, polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, agarose, cellulose, nitrocellulose, dextran, sephadex, sepharose, liposome, carboxymethyl cellulose, polyacrylamide, polyesterine, gabbro, filter paper, ion exchange resin, plastic film, plastic tube, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, metal, glass, glass beads, or magnetic particles. Other solid substrates include cell culture plates, ELISA plates, tubes, and polymeric membranes. The support may have any possible shape, for example spherical (bead), cylindrical (inside a test tube or well), planar (sheet, test strip).

[0148] Labels capable of generating a detectable signal enable qualitative or quantitative measurement of the formation of antigen-antibody complexes, and examples of such labels include enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, and radioactive isotopes. Enzymes that can be used include β-glucuronidase, β-D-glucosidase, urease, peroxidase (such as horseradish peroxidase), alkaline phosphatase, acetylcholinesterase, glycose oxidase, hexokinase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, invertase, and luciferase. Fluorescent substances that can be used include fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, and fluorsine isothiocyanate. Ligands include biotin derivatives, and luminescent substances include acridinium esters and luciferin. Microparticles include colloidal gold and colored latex, and redox molecules include ferrocene, ruthenium complexes, viologen, quinone, Ti ion, Cs ion, diimide, 1,4-benzoquinone, and hydroquinone. Radioisotopes include 3 H, 14 C, 32 P, 35 S, 36 Cl, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, 186 Re, etc. However, in addition to those exemplified above, any that can be used in immunological analysis can be used.

[0149] As an enzyme chromogenic substrate, for example, when horseradish peroxidase (HRP) is selected as an enzyme label, a solution containing 3-amino-9-ethylcarbazole, 5-aminosalicylic acid, 4-chloro-1-naphthol, o-phenylenediamine, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid), 3,3-diaminobenzidine, 3,3',5,5'-tetramethylbenzidine, o-dianisidine, or 3,3-dimethoxybenzidine can be used as a substrate. In addition, when alkaline phosphatase is selected as an enzyme label, a solution containing 5-bromo-4-chloro-3-indolyl phosphate, nitroblue tetrazolium, or p-nitrophenyl phosphate can be used as a substrate. Additionally, when β-D-galactosidase is selected as an enzyme marker, a solution containing o-nitrophenyl-β-D-galactoside or 5-bromo-4-chloro-3-indole-β-D-galactopyranoside can be used as a substrate. In addition, various enzymes and enzyme chromogenic substrates known in the art can be used.

[0150] A method for detecting FAM167A according to one embodiment of the present invention may detect the FAM167A protein in a sample or an animal model other than a human. Specifically, the method for detecting FAM167A according to the present invention may include a step of detecting a FAM167A antigen-antibody complex using the aforementioned monoclonal antibody or an antigen-binding fragment thereof.

[0151] In the present invention, the sample is a biological sample, and may include, but is not limited to, tissue, cell, whole blood, serum, plasma, tissue autopsy samples (brain, skin, lymph node, spinal cord, etc.), cell culture supernatant, ruptured eukaryotic cells, and bacterial expression systems. For example, the biological sample may be isolated from a mammal, including a human, having a disease in which FAM167A is overexpressed or a disease related to FAM167A, or may be isolated from an animal model that overexpresses the FAM167A protein or an animal model of a disease related to FAM167A, but is not limited thereto. The presence or absence of the FAM167A protein can be confirmed by reacting these biological samples with or without manipulation with the monoclonal antibody of the present invention or an antigen-binding fragment thereof.

[0152] In the present invention, the animal model may be an animal model that overexpresses the FAM167A protein or a specific disease related to FAM167A.

[0153] The above animal model is a mammal other than a human, including, but not limited to, livestock (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats). Preferably, it may be a rodent, and more preferably, a mouse.

[0154] In one embodiment of the present invention, the disease associated with FAM167A may be, but is not limited to, chronic myeloid leukemia with BCR-ABL-independent tyrosine kinase inhibitor (TKI) resistance.

[0155] The fifth aspect of the present invention relates to an epitope of a mouse FAM167A antigen consisting of an amino acid sequence of SEQ ID NO: 18 and a nucleic acid molecule encoding the same.

[0156] In relation to the fifth aspect, the present invention provides the use of an epitope of a mouse FAM167A antigen consisting of the amino acid sequence of SEQ ID NO: 18 for the production of a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to FAM167A according to the first aspect described above.

[0157] The epitope of the mouse FAM167A antigen according to one embodiment of the present invention is a site to which the monoclonal antibody or antigen-binding fragment thereof that specifically binds to FAM167A according to the first aspect described above specifically binds, and may be composed of amino acid residues of SEQ ID NO: 18 (MSVPQIQVEEVSEKDRPAGAAVPPD) corresponding to positions 1 to 25 of the full-length amino acid sequence of the mouse FAM167A protein (e.g., the amino acid sequence of SEQ ID NO: 17).

[0158] In the present invention, the nucleic acid molecule encoding the epitope may include, without limitation, a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 18. The nucleic acid molecule is interpreted to also include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence described above. The substantial identity refers to a nucleotide sequence that exhibits at least 80% identity, more preferably at least 90% identity, when the nucleotide sequence of the present invention described above and any other sequence are arranged to correspond as much as possible and the arranged sequence is analyzed using an algorithm commonly used in the art.

[0159] Hereinafter, the present invention will be described in detail by way of examples. However, the following examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following examples.

[0160] [Example 1]

[0161] Production and sequence analysis of a monoclonal antibody for detecting mouse FAM167A.

[0162] A monoclonal antibody was manufactured by IntoApp Co., Ltd. that binds to a fragment consisting of amino acids 1 to 25 (SEQ ID NO: 18) of the full-length mouse FAM167A protein. The antibody was manufactured using a phage display library. The expression information of the final selected anti-mFam167a-1-BSA antibody is shown in Table 2.

[0163] Source: 293F cell culture volume: 50 ml. Form: Minibody (scFv-Fc fusion protein). Composition: Human scFv + human IgG1-Fc.

[0164] The purification method and purity of the expressed antibody are shown in Table 3. The purity of the purified anti-FAM167A monoclonal antibody was analyzed using SDS-PAGE and Commassie Brilliant Blue (CBB) staining. The analysis results are shown in Figure 1 and Table 3.

[0165] Affinity chromatography (resin) Protein G agarose (GE) purity >95% (as measured by SDS-PAGE and CBB staining)

[0166] As confirmed in Figure 1 and Table 3, only proteins within the expected protein size range were detected, and the anti-FAM167A monoclonal antibody (minibody) was purified to a purity of >95%. The amino acid sequence of the purified anti-FAM167A monoclonal antibody was analyzed by the manufacturer, IntoApp Co., Ltd. The analysis results are shown in Tables 4 to 6.

[0167] Amino acid sequence SEQ ID NO: Light chain CDR1 SSNIGSNY1 Light chain CDR2 KNN2 Light chain CDR3 AAWDDSLSGWV3 Heavy chain CDR1 GGAFSTSA4 Heavy chain CDR2 VIPVLGTV5 Heavy chain CDR3 ARERAYGSATGLAH6

[0168] 아미노산 서열서열번호경쇄 가변 영역ELVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYKNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTELTVLG7중쇄 가변 영역QVQLVQSGAEVKEPGSSVRVSCKVSGGAFSTSALTWVRQAPGQGLQWLGRVIPVLGTVNIAQKFEDRFTIKADESTSTVYMELSDMRRDDTAVYFCARERAYGSATGLAHWGQGTLVTVSS8링커GGSSRSSSSGGGGSGGGG9중쇄 불변 영역의 CH1 단편ASPTSPKVTS10미니바디 전장ELVLTQPPSASGTPGQRVTISCSGSSSNIGSNYVYWYQQLPGTAPKLLIYKNNQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGWVFGGGTELTVLGGGSSRSSSSGGGGSGGGGQVQLVQSGAEVKEPGSSVRVSCKVSGGAFSTSALTWVRQAPGQGLQWLGRVIPVLGTVNIAQKFEDRFTIKADESTSTVYMELSDMRRDDTAVYFCARERAYGSATGLAHWGQGTLVTVSSASPTSPKVTS11

[0169] 뉴클레오타이드 서열서열번호경쇄 가변 영역GAGCTCGTGCTGACTCAGCCACCTTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATTATGTATACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAAGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCCGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAGTGGTTGGGTGTTCGGCGGAGGGACCGAGCTGACCGTCCTCGGC12중쇄 가변 영역CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGGAGCCTGGGTCCTCGGTAAGAGTCTCCTGCAAGGTCTCTGGAGGCGCCTTCAGCACCTCTGCTCTCACCTGGGTGCGACAGGCCCCTGGACAAGGCCTTCAGTGGCTGGGGAGGGTCATCCCTGTTCTTGGAACAGTTAACATCGCACAGAAATTCGAGGACAGATTCACCATTAAAGCGGACGAATCCACAAGCACAGTCTACATGGAGTTGAGCGACATGAGGCGCGACGACACGGCCGTCTATTTTTGCGCGCGAGAGAGGGCTTATGGATCGGCAACGGGTTTGGCCCACTGGGGCCAGGGAACACTGGTCACCGTCTCCTCA13링커GGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGCTCGGGCGGTGGTGGG14중쇄 불변 영역 CH1 단편GCATCCCCGACCAGCCCCAAGGTCACTAGT15미니바디전장GAGCTCGTGCTGACTCAGCCACCTTCAGCGTCTGGGACCCCCGGGCAGAGGGTCACCATCTCTTGTTCTGGAAGCAGCTCCAACATCGGAAGTAATTATGTATACTGGTACCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAAGAATAATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCCGGCACCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAGTGGTTGGGTGTTCGGCGGAGGGACCGAGCTGACCGTCCTCGGCGGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGCTCGGGCGGTGGTGGGCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGGAGCCTGGGTCCTCGGTAAGAGTCTCCTGCAAGGTCTCTGGAGGCGCCTTCAGCACCTCTGCTCTCACCTGGGTGCGACAGGCCCCTGGACAAGGCCTTCAGTGGCTGGGGAGGGTCATCCCTGTTCTTGGAACAGTTAACATCGCACAGAAATTCGAGGACAGATTCACCATTAAAGCGGACGAATCCACAAGCACAGTCTACATGGAGTTGAGCGACATGAGGCGCGACGACACGGCCGTCTATTTTTGCGCGCGAGAGAGGGCTTATGGATCGGCAACGGGTTTGGCCCACTGGGGCCAGGGAACACTGGTCACCGTCTCCTCAGCATCCCCGACCAGCCCCAAGGTCACTAGT16

[0170] [실시예 2]

[0171] ELISA를 이용한 미니바디 활성 측정

[0172] The target (1 μg / ml) diluted in coating solution was added to a high binding 96-well plate (Cat. No. 3690) and incubated at 4°C. After washing the wells with washing solution, blocking solution was added and incubated at room temperature. After washing the wells with washing solution, minibody (anti-mFam167a-1-BSA antibody, 2 μg / ml) was added as the primary Ab and incubated at room temperature. After washing the wells with washing solution, anti-human IgG-HRP (1:20000) was added as the secondary Ab and incubated at room temperature. After washing the wells with washing solution, TMB substrate was added and incubated at room temperature for 5 minutes, and minibody activity was measured using a plate reader. Specific ELISA conditions and experimental results are shown in Tables 7 and 8, respectively.

[0173] Fixed Corning (Cat. No. 3690) Target mFam167a-1-BSA (1 μg / ml), negative (BSA 1 μg / ml) Coating solution Sodium bicarbonate buffer (pH 9.6) Blocking solution 3% M-PBST Washing solution PBST Primary Ab 2 μg / ml Secondary Ab-HRP 1:20,000 TMB Substrate Incubation time 5 min Measurement filter 450 nm Plate reader Epoch, BIOTEK

[0174] Anti-mFam167a-1-BSA (2T1A10) minibody 2 μg / ml Antigen (mFam167a-1-BSA) BSA background control 1 μg / ml 1 μg / ml 3.5 1 1 0.06 6 0.04 5 3.5 0 0.06 7 0.04 6

[0175] As confirmed in Table 8, the minibody was shown to specifically bind to the mFam167A antigen.

[0176] [Example 3]

[0177] Confirmation of binding of FAM167A to minibody using Western blot

[0178] HEK293T cells were transfected with pMigR1-Empty (Addgene Plasmid #27490), pMigR1-hFam167a overexpression vector, and pCMV6-mFam167a overexpression vector (Origene CAT#: MR202374) using Lipofectamine 3000. The pMigR1-hFam167a overexpression vector was constructed by inserting the nucleotide sequence of human Fam167a (SEQ ID NO: 19) disclosed in NCBI Reference Sequence: NM_053279.3 into pMigR1-Empty (Addgene Plasmid #27490).

[0179]

[0180]

[0181] Each cell was placed in 2Y sampling buffer [2 mL Tris (1 M, pH 6.8), 4.6 mL glycerol (50%), 1.6 mL SDS (10%), 0.4 mL bromophenol blue (0.5%), 0.4 mL β-mercaptoethanol)] and heated to 100°C. Afterwards, centrifugation was performed at 13,200 rpm for 10 minutes to isolate only the supernatant.

[0182] Samples were loaded onto a 12.5% ​​acrylamide gel, and proteins were separated by size using SDS-PAGE (Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis) at 120 V. The separated proteins on the 12.5% ​​acrylamide gel were transferred to a PVDF transfer membrane (Cat. No. BSP0161) at 200 mA overnight. The PVDF transfer membrane was blocked with skim milk (5%, TTBS base) and washed with TTBS buffer (20 mM Tris, 150 mM NaCl, 0.1% Tween® 20 detergent, pH 7.6). The primary antibody (anti-GAPDH antibody, or minibody, or anti-Myc tag antibody) (1:1000 dilution, 3% BSA, TTBS base) was bound, and washed with TTBS buffer. Secondary ab (anti-mouse IgG-HRP, anti-human IgG-HRP, same as ELISA) (1:10000 dilution, 3% skimmed milk, TTBS base) was bound and washed with TTBS buffer. The PVDF transfer membrane was treated with an ECL kit (RPN2235, RPN2232), and the PVDF transfer membrane was photographed using LAS4000 (GE Healthcare, ImageQuant).

[0183] As shown in Fig. 2, the expression locations of mouse Fam167a detected using anti-Myc tag antibodies and minibody, respectively, were identical, confirming that the minibody specifically binds to mouse Fam167a.

[0184] [Example 4]

[0185] Confirmation of binding of FAM167A to minibodies using flow cytometry

[0186] HEK293T cells were transfected with pMigR1-Empty, pMigR1-hFam167a overexpression vector, and pCMV6-mFam167a overexpression vector using Lipofectamine 3000. Each cell was treated with Foxp3 / Transcription Factor Fixation / Permeabilization buffer (Cat. No. 00-5521-00). Staining was performed using anti-Myc tag antibody (Cat. No. 2279S) (Fig. 3), minibody and secondary ab (anti-human IgG-Alexa Fluor™ 488) (Fig. 4), Alexa Fluor™ 594 antibody labeling kit (Cat. No. A20185) or Alexa Fluor™ 488 antibody labeling kit (Cat. No. A20181) (Fig. 5), and minibody conjugated with anti-Myc tag antibody and Alexa Fluor™ 594 or Alexa Fluor™ 488 fluorescence (Fig. 6). After staining, the cells were washed with PBS (Phosphate-Buffered Saline buffer), and then flow cytometry analysis was performed using Cytoflex (Beckman Coulter).

[0187] The results of flow cytometry analysis are shown in Figures 3 to 6. As shown in Figure 3, the expression of mouse Fam167a overexpressed in HEK293T cells was confirmed through an anti-Myc tag antibody. In addition, as shown in Figures 4 to 6, the minibody was confirmed to specifically bind to HEK293T cells overexpressing mouse Fam167a through a minibody conjugated with a secondary ab (anti-human IgG-Alexa Fluor™ 488) and fluorescence.

[0188] This patent application is the result of research conducted with the support of the National Research Foundation of Korea (① Project No.: 2021R1A2C3011211, Research Project Title: Study on the Role of Gammaherpes Virus Infection in Neuroinflammatory Diseases, Research Period: 2023.03.01 ~ 2024.02.29; ② Project No.: 2022M3A9I2017587, Research Project Title: Development of a Multi-Omics-Based Future New and Variant Influenza Mutant Response Platform, Research Period: 2023.01.01 ~ 2023.12.31) funded by the Government of the Republic of Korea (Ministry of Science and ICT).

Claims

1. A monoclonal antibody or antigen-binding fragment thereof that specifically binds to FAM167A, comprising a light chain variable region comprising a light chain CDR1 consisting of an amino acid sequence of SEQ ID NO: 1, a light chain CDR2 consisting of an amino acid sequence of SEQ ID NO: 2, and a light chain CDR3 consisting of an amino acid sequence of SEQ ID NO: 3, and a heavy chain variable region comprising a heavy chain CDR1 consisting of an amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 consisting of an amino acid sequence of SEQ ID NO: 5, and a heavy chain CDR3 consisting of an amino acid sequence of SEQ ID NO:

6.

2. In the first paragraph, the monoclonal antibody is a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to FAM167A, comprising a light chain variable region consisting of an amino acid sequence of SEQ ID NO: 7 and a heavy chain variable region consisting of an amino acid sequence of SEQ ID NO:

8.

3. In the first paragraph, the monoclonal antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to FAM167A, which is a mouse antibody, chimeric antibody, humanized antibody, or fully human antibody.

4. In the first paragraph, the monoclonal antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to FAM167A, selected from the group consisting of whole IgG, Fab, Fab', F(ab')2, xFab, scFab, dsFv, Fv, scFv, scFv-Fc, scFab-Fc, diabody, minibody, scAb, dAb, half-IgG, and combinations thereof.

5. In the first paragraph, the monoclonal antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to FAM167A, wherein a linker consisting of an amino acid sequence of SEQ ID NO: 9 is connected between a light chain variable region and a heavy chain variable region.

6. In the first paragraph, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant C consisting of an amino acid sequence of SEQ ID NO:

10. H A monoclonal antibody or antigen-binding fragment thereof that specifically binds to FAM167A, further comprising a fragment.

7. In the first paragraph, the monoclonal antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to FAM167A, which is a minibody consisting of the amino acid sequence of SEQ ID NO:

11.

8. In paragraph 1, the monoclonal antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to FAM167A, which is used for flow cytometry analysis, Western blot analysis, or ELISA (Enzyme Linked Immunosorbent Assay) analysis.

9. In the first paragraph, the monoclonal antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to FAM167A and binds to an epitope consisting of the amino acid sequence of SEQ ID NO:

18.

10. A nucleic acid molecule encoding a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9.

11. A nucleic acid molecule according to claim 10, wherein the nucleic acid molecule encoding the light chain of the monoclonal antibody or antigen-binding fragment thereof comprises a nucleotide sequence of SEQ ID NO: 12, and the nucleic acid molecule encoding the heavy chain of the monoclonal antibody or antigen-binding fragment thereof comprises a nucleotide sequence of SEQ ID NO:

13.

12. In the 10th paragraph, when the monoclonal antibody or antigen-binding fragment thereof is a minibody, the nucleic acid molecule encoding the minibody is a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO:

16.

13. A vector comprising the nucleic acid molecule of Article 10.

14. A host cell containing the vector of clause 13.

15. A method for producing a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to FAM167A, comprising a step of culturing the host cell of item 14.

16. A kit for detecting FAM167A, comprising a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9.

17. A kit for detecting FAM167A, wherein the kit is an ELISA (Enzyme Linked Immunosorbent Assay) kit in the 16th paragraph.

18. A method for detecting FAM167A protein in a sample or an animal model other than a human, comprising a step of detecting a FAM167A antigen-antibody complex using a monoclonal antibody or an antigen-binding fragment thereof of any one of claims 1 to 9.

19. An epitope of the mouse FAM167A antigen consisting of the amino acid sequence of sequence number 18.

20. A nucleic acid molecule encoding the epitope of item 19.

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