Anti-APP antibody or antigen-binding fragment thereof, and uses thereof

Abstract

The present invention relates to an anti-APP antibody or an antigen-binding fragment thereof, and uses thereof. Specifically, the anti-APP antibody or antigen-binding fragment thereof according to the present invention may be utilized for analysis and detection of cell membrane APP, and further for diagnosis of APP-related diseases.

Description

Anti-APP antibodies or their antigen-binding fragments, and their uses

[0001] The present invention relates to an anti-APP (amyloid-beta precursor protein) antibody or an antigen-binding fragment thereof, and uses thereof. Specifically, the present invention relates to an anti-APP antibody or an antigen-binding fragment thereof for analyzing or detecting the expression of APP in a cell membrane.

[0002] This application claims the benefit of priority to Korean Patent Application No. 10-2025-0039823 filed on March 27, 2025, the entire contents of which are incorporated herein by reference.

[0003] Flow cytometry is a method for characterizing cells by measuring factors such as gene expression or protein levels. Through this, it is possible to understand the function, state, and changes of cells and gain insights into the onset and progression of diseases. In particular, fluorescent antibodies can be used to quantify and classify the expression levels of membrane proteins in living cells.

[0004] APP belongs to the type I transmembrane protein family and is expressed in three isoforms—APP695, APP751, and APP770—through alternative splicing. APP695 is primarily expressed in neurons, whereas APP751 and APP770 are expressed mostly in tissues and contain a 56-amino acid Kunitz protease inhibitor (KPI) domain within the extracellular region. APP is best known for releasing amyloid-generating Aβ peptides, which are cleaved by β-secretase and subsequently by γ-secretase to form amyloid plaques in the brains of Alzheimer's disease patients. Several studies have reported that the soluble APP outer domain and APP intracellular domain produced at this stage also impair normal APP function and contribute to disease progression. Furthermore, APP has been reported to function as a cell surface receptor through interactions with proteins such as APLP2, N-Cadherin, and NgCAM, contributing to cell-cell and cell-matrix interactions necessary for neurite growth, dendritic branching, and synaptic adhesion. Recently, it has been revealed that APP can inhibit T cell activity by binding to CNTN4 (Contactin-4), an immune checkpoint protein highly expressed in various tumor tissues, and by being expressed on the T cell membrane, attenuating T cell receptor signals and reducing intercellular adhesion.

[0005] However, to support the claim that APP acts as a cell surface receptor to perform physiological roles, it is necessary to be able to analyze or detect the expression of cell membrane APP; while fluorescently labeled anti-APP antibodies are required for this purpose, there are currently no commercially available FACS antibodies.

[0006] The object of the present invention is to provide an anti-APP antibody that specifically binds to APP or an antigen-binding fragment thereof.

[0007] Specifically, we intend to provide an anti-APP antibody or an antigen-binding fragment thereof for analyzing or detecting APP expressed in the cell membrane.

[0008] [1] The present invention comprises a heavy chain CDR 1 having the amino acid sequence of SEQ ID NO. 3;

[0009] Heavy chain CDR 2 containing the amino acid sequence of SEQ ID NO. 4;

[0010] Heavy chain CDR3 containing the amino acid sequence of SEQ ID NO. 5;

[0011] Light chain CDR 1 containing the amino acid sequence of SEQ ID NO. 6;

[0012] Light chain CDR 2 comprising the amino acid sequence of SEQ ID NO. 7; and

[0013] Light chain CDR 3 containing the amino acid sequence of SEQ ID NO. 8;

[0014] The invention relates to an anti-APP (Amyloid-beta precursor protein) antibody or an antigen-binding fragment thereof comprising

[0015] [2] The present invention relates to an anti-APP antibody or an antigen-binding fragment thereof comprising a heavy chain variable region of SEQ ID NO. 1 and a light chain variable region of SEQ ID NO. 2.

[0016] [3] In the above [1] or [2], the anti-APP antibody or its antigen-binding fragment is 1 X 10 -8 Equilibrium dissociation constant less than or equal to M (K D It can be combined with APP using ).

[0017] [4] In the above [1] or [2], the anti-APP antibody or its antigen-binding fragment is 100 nM or less EC 50It can be combined with the APP.

[0018] [5] In [1] or [2] above, the anti-APP antibody or its antigen-binding fragment is Fab, Fab', Fab'-SH, Fv, single-strand antibody scFv, F(ab')2 fragment, VL, VH, diabody, triabody, tetrabody, minibody ((scFV-CH3)2), IgG-delta CH2, scFv-Fc, (scFv)2-Fc, Fynomer, FynomAbs (fynomers fused to antibodies), dual-affinity re-targeting (DART), AlbudAbs, BiTEs (bispecific T-cell engager), TandAbs (tandem diabodies), DAFs (dual acting Fab), two-in-one antibodies, SMIPs (small modular immunopharmaceuticals), anticalins, FN3 It may be a monobody, DARPins, Affibodies, Affilins, Affimers, Affitins, Alphabodies, Avimers, Im7, VLR, VNAR, Trimab, CrossMab, TRIDENT, a nanobody, binanobodies or D-sdFv, DVD-Igs (dual variable domain immunoglobulin), CovX-bodies (peptide modified antibodies), a duobody or triomAbs.

[0019] [6] In the above [1] or [2], the anti-APP antibody or its antigen-binding fragment may be a chimeric antibody, a humanized antibody, or a human antibody.

[0020] [7] The present invention relates to an antibody conjugate comprising the anti-APP antibody of [1] or [2] or an antigen-binding fragment thereof and a detectable label.

[0021] [8] The present invention relates to a polynucleotide encoding the anti-APP antibody of [1] or [2] or the antigen-binding fragment thereof.

[0022] [9] The present invention relates to a recombinant expression vector comprising the polynucleotide of [8].

[0023]

[0010] The present invention relates to a composition for analyzing or detecting the expression of a cell membrane APP, comprising the anti-APP antibody of [1] or [2] or an antigen-binding fragment thereof.

[0024]

[0011] The present invention relates to a composition for diagnosing APP-related diseases comprising the anti-APP antibody of [1] or [2] or an antigen-binding fragment thereof.

[0025]

[0012] In the above

[0011] , the APP-related disease may be cancer, Alzheimer's disease, Creutzfeldt-Jakob disease, multiple sclerosis, Parkinson's disease, Down syndrome, amyloidosis, or polyneuropathy.

[0026]

[0013] The present invention relates to a method for providing information on APP-related diseases, comprising the step of measuring the cell membrane APP expression level in cells isolated from a cultured cell line or individual using the anti-APP antibody of [1] or [2] or the antigen-binding fragment thereof.

[0027]

[0014] In the above

[0013] , the APP-related disease may be cancer, Alzheimer's disease, Creutzfeldt-Jakob disease, multiple sclerosis, Parkinson's disease, Down syndrome, amyloidosis, or polyneuropathy.

[0028]

[0015] The present invention relates to a kit for diagnosing APP-related diseases comprising the composition of

[0011] .

[0029]

[0016] The present invention relates to the use of the anti-APP antibody of [1] or [2] or its antigen-binding fragment for analyzing or detecting the expression of cell membrane APP in cells isolated from a cultured cell line or individual.

[0030]

[0017] The present invention relates to the use of the anti-APP antibody of [1] or [2] or its antigen-binding fragment for diagnosing APP-related diseases.

[0031]

[0018] In the above

[0017] , the APP-related disease may be cancer, Alzheimer's disease, Creutzfeldt-Jakob disease, multiple sclerosis, Parkinson's disease, Down syndrome, amyloidosis, or polyneuropathy.

[0032] The novel anti-APP antibody or its antigen-binding fragment of the present invention binds to APP proteins with high affinity. Through this, the anti-APP antibody or its antigen-binding fragment of the present invention can be usefully used to analyze or detect the expression of APP in the cell membrane.

[0033] Figure 1 is a diagram showing the SDS-PAGE analysis results of M001A, a type of anti-APP antibody of the present invention.

[0034] FIG. 2 is a chromatogram of the SEC-HPLC analysis results of M001A, a type of anti-APP antibody of the present invention (horizontal axis: time (Minutes), vertical axis: distance (AU)).

[0035] Figure 3 is a chromatogram of the SEC-HPLC analysis results of M001A-AF647, a type of anti-APP antibody-fluorescent conjugate of the present invention (horizontal axis: time (Minutes), vertical axis: distance (AU)).

[0036] Figure 4 is a diagram showing data confirming the degree of binding of M001A to human APP protein as absorbance at 450 nm according to concentration (nM) through an ELISA experiment.

[0037] Figure 5 is a diagram showing data confirming the degree of binding of M001A to HEK293 cells overexpressing human APP protein through FACS experiments using the gMFI (Geometric Mean Fluorescence Intensity) value of allophycocyanin (APC) according to concentration (nM).

[0038] Figure 6 is a diagram showing data confirming the ability of M001A to inhibit the interaction between human APP protein and human CNTN4 protein through a competitive ELISA experiment, expressed as the degree of APP / CNTN4 binding at 450 nm according to concentration (nM) (APP / CNTN4 binding, %).

[0039] Figure 7 is a diagram showing the presence or absence of App protein expression in the spleens of wild-type (WT) and knockout (KO) mice isolated (Figure 7a) and the percentage of App protein expression induced by anti-CD3 antibody stimulation on CD4 and CD8 T cell membranes using M001A-AF647 (Figure 7b).

[0040] Figure 8 is a figure showing APP mRNA expression (Figure 8a) and cell membrane APP protein expression (Figure 8b) using M001A-AF647 in % after isolating human CD4 and CD8 T cells and stimulating them with an anti-CD3 antibody.

[0041] The present invention will be described in detail below.

[0042] definition

[0043] In this specification, the term “consisting of” means that the proportion of a specific component(s) is 100% of the total. The component or feature following the term “consisting of” may be essential or mandatory.

[0044] In this specification, the term "comprising" means the presence of the features, steps, or components described below the term, and does not exclude the presence or addition of one or more features, steps, or components. In this specification, the components or features described below "comprising" may be essential or mandatory, but some embodiments may further include other optional or non-essential components or features.

[0045] In this specification, the term "comprising" may be modified to refer to "consisting essentially of" or "consisting of" in some embodiments.

[0046] In this specification, the term “complementarity determining region” or “Complementarity determining region, CDR” refers to amino acid residues in an antibody variable region that are present for antigen binding. Each variable region typically has three CDR regions identified as CDR1, CDR2, and CDR3. The CDR contains most of the residues that cause specific interactions between the antibody (or its antigen-binding fragment) and the antigen, and thus contribute to the functional activity of the antibody molecule. These are the primary determining regions of antigen specificity.

[0047] In this specification, the term “variable region” refers to a specific portion of the heavy and light chains of an antibody molecule comprising the amino acid sequences of the antibody’s CDR and framework region (FR).

[0048] In this specification, the term “antibody” is an immunoglobulin molecule capable of specifically binding to a target, e.g., carbohydrates, polynucleotides, lipids, polypeptides, proteins, etc., through at least one antigen recognition site located within the variable region of the immunoglobulin molecule. The term “antibody” in this specification is used in the broadest sense and is therefore intended to include not only intact polyclonal or monoclonal antibodies, but also dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), their antigen-binding fragments, antibody fragments, fusion proteins comprising any other modified arrangement of an immunoglobulin molecule including an antigen recognition site (e.g., variable region), synthetic antibodies (e.g., antibody mimics), FynomAbs, etc.

[0049] In this specification, the term "multispecific antibody" refers to an antibody comprising a plurality of variable regions, each variable region having binding specificity for different epitopes. Types thereof include bispecific antibodies or trispecific antibodies.

[0050] There are five types of antibodies: immunoglobulin (Ig)M, IgD, IgG, IgA, and IgE, each containing a heavy chain produced from the heavy chain constant region genes μ, δ, γ, α, and ε. The light and heavy chains of antibodies are divided into a variable region, where the amino acid sequence differs for each antibody, and a constant region, where the amino acid sequence is the same. The heavy chain constant region contains CH1, H(hinge), CH2, and CH3 domains. Each domain consists of two β-sheets connected by an intramolecular disulfide bond.

[0051] As used herein, the terms “antigen-binding fragment” or “antibody fragment” typically include not only at least a portion (e.g., one or more CDRs) or variable regions of the antigen-binding domain of a parent antibody, but also any modified form including a specific recognition site for a target antigen, e.g., other modified arrangement forms of an immunoglobulin molecule including a specific antigen recognition site, such as glycosylated variants of antibodies, amino acid sequence variants of antibodies, covalently modified antibodies, and antigen-binding antibodies thereof.

[0052] As used herein, the terms “to bind specifically to” or “specific to” mean having deterministic power regarding the presence of a target in the presence of a heterogeneous population of molecules including biological molecules, and refer to measurable and reproducible interactions such as binding between a target and an antibody. For example, an antibody that binds specifically to a specific target (e.g., an epitope) means an antibody that binds to said target with greater affinity, greater binding strength, more readily, and / or for a longer duration than it binds to other targets.

[0053] In this specification, the term "K D " refers to the binding equilibrium dissociation constant of a specific antibody-antigen interaction, and K D It is calculated through the formula = Kd / Ka (where Ka is the bonding rate constant and Kd is the dissociation rate constant), and the above constant K D has units of M. K for antibodies D The value can be measured using methods widely established in the industry. The antibody's K D Specific methods for measuring values ​​may include surface plasmon resonance (SPR), more specifically using a biosensor system, for example, the Biacore® system, or bio-layer interferometry (BLI), for example, the Octet® system.

[0054] In this specification, the term "EC" 50 " is a term related to in vitro or in vivo analysis using antibodies, meaning the concentration of antibody that induces 50% of the maximum response, that is, the intermediate response between the maximum response and the baseline.

[0055] In this specification, the term “Fab fragment” refers to a monovalent fragment consisting of VL, VH, CL, and CH1 domains.

[0056] In this specification, the term “Fab’” fragment refers to a monovalent fragment in which several residues are added to the carboxyl terminus of a CH1 domain containing one or more cystes from an antibody hinge region.

[0057] In this specification, the term "Fab'-SH" refers to Fab' in which a cysteine ​​residue of the invariant domain possesses a free thiol group.

[0058] In this specification, the term "F(ab')2 antibody fragment" refers to a pair of Fab' fragments formed through hinge cysteine ​​between the Fab' fragments.

[0059] In this specification, the term "Fv" refers to a minimal antibody fragment containing a complete antigen recognition site and an antigen binding site. This fragment consists of a dimer in which one heavy chain variable region and one light chain variable region are tightly non-covalently associated. These two regions fold to form six hypervariable loops (three loops each from the heavy and light chains), said loops providing amino acid residues for antigen binding and conferring antigen binding specificity to the antibody. However, even a single variable region possesses the ability to recognize and bind to an antigen, although its affinity is lower than that of the entire binding site.

[0060] In this specification, the term “single-chain antibody scFv” is an antibody fragment comprising VH and VL antibody domains linked by a single polypeptide chain. The “single-chain antibody scFv” may further comprise a polypeptide linker between the VH and VL domains so that the scFv can form a structure intended for antigen binding. In this specification, it may be referred to as the scFv antibody fragment, antigen-binding fragment scFv, scFv antibody, antibody scFv, or simply scFv.

[0061] In this specification, the term “diabody” refers to a small antibody fragment produced by constructing a scFv fragment using a short linker (about 5-10 residues) between the VH and VL domains so that intra-chain rather than inter-chain pairing of the V domain is achieved to produce a divalent fragment, that is, a fragment having two antigen-binding sites.

[0062] In this specification, the term “bispecific diabody” is a heterodimer composed of two “cross-linked” scFv fragments in which the VH domains and VL domains of two antibodies are located on different polypeptide chains. Similarly, triabody and tetrabody each comprise three polypeptide chains and four polypeptide chains, respectively, and form three antigen-binding sites and four antigen-binding sites, respectively, which may be identical or different.

[0063] In this specification, the term "Fynomer" refers to a non-immunoglobulin-derived conjugated polypeptide derived from a human Fyn SH3 domain. Fyn SH3-derived polypeptides are well known in the art and are mentioned, for example, in the literature [Grabulovski et al. (2007) JBC, 282, p. 3196-3204], WO 2008 / 022759, etc. Fynomers can be genetically engineered to fuse with other molecules (e.g., antibodies) to produce "FynomAbs," which can be processed to have bispecificity, for example.

[0064] In this specification, the term “dual-affinity re-targeting (DART) or TRIDENT” refers to a dual-specific antibody platform designed to bind to two or more targets simultaneously. DART refers to a covalently linked dual-specific diabody, for example, a diabody linked via a C-terminal disulfide bridge, and its specific structure and definition are described in the literature [J. Mol. Biol. (2010) 399, 436-449], etc.

[0065] In this specification, the term "chimeric antibody" means an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, for example, an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody. Methods for producing chimeric antibodies are known in the art. For example, reference may be made to the literature [Morrison, Science 229:1202 (1985)], the entirety of which is incorporated herein by reference.

[0066] In this specification, the term “humanized antibody” means an antibody in which one or more CDR sequences derived from a non-human species, e.g., other mammalian species such as mouse or chicken germlines, are inserted into a framework sequence from a human immunoglobulin molecule. The framework sequence may be further modified, e.g., by mutation. Reference to the human immunoglobulin sequence may be made, e.g., to the NCBI Database (Entez Gene). By using a suitable sequence, the immunogenicity of the antibody may be reduced, or binding affinity, affinity, on-rate, off-rate, affinity, specificity, half-life, or any other suitable feature may be reduced, enhanced, or altered.

[0067] In this specification, the term "human antibody" refers to an antibody comprising a variable region in which both the framework and the CDR region are derived from a human immunoglobulin sequence. The constant region of the antibody is also derived from a human immunoglobulin sequence.

[0068] In this specification, the term "antibody conjugate" refers to an anti-APP antibody of the present invention or an antigen-binding fragment thereof bound to another substance, e.g., a detectable label. For example, "antibody-fluorescent conjugate" refers to an anti-APP antibody of the present invention or an antigen-binding fragment thereof bound to a fluorescent substance.

[0069] In this specification, the term “detectable label” may be any molecule capable of producing a signal or being induced to produce a signal, including but not limited to fluorescent substances (e.g., fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, red fluorescent protein, yellow fluorescent protein, etc.), radioactive labels (e.g., 3H, 125I, 35S, 14C, or 32P), enzymes (e.g., mustard peroxidase, alkaline phosphatase, luciferase, and enzymes commonly used in enzyme-linked immunosorbent assays (ELISA)), chemiluminescent substances, or photosensitizers. Accordingly, the anti-APP antibody of the present invention or its antigen-binding fragment may be detected and / or measured by detecting fluorescence or luminescence, radioactivity, enzymatic activity, or light absorption, and said detectable label may be attached to the anti-APP antibody of the present invention or its antigen-binding fragment by conventional methods known in the art.

[0070] In this specification, the term “operably combined” means a case where two DNA fragments are combined such that the amino acid sequence encoded by the two DNA fragments remains in an in-frame state.

[0071] In this specification, the term "vector" refers to a DNA molecule capable of self-replicating in prokaryotic and / or eukaryotic cells, used interchangeably with recombinant vectors, cloning vectors, and expression vectors, and is generally used as an intermediate carrier for delivering genes or DNA fragments to cells, etc. A vector typically comprises, but is not limited to, a replication origin capable of replicating in prokaryotic and / or eukaryotic cells, a selectable marker gene capable of conferring resistance to specific conditions / substances such as antibiotic-degrading enzymes, a promoter capable of gene transcription in eukaryotic or prokaryotic cells, and a translatable sequence.

[0072] In this specification, the term "plasmid" refers to a circular double standard DNA loop to which additional DNA fragments can be conjugated, and corresponds to a type of vector. Another type of vector is a viral vector to which additional DNA fragments can be conjugated to a viral genome. Certain vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors having a bacterial replication origin and episomal mammalian vectors).

[0073] In this specification, the term "phage library" refers to a technique for producing an antibody library in which, for example, the genes for the heavy and light chain variable regions of a human antibody are fused to a phage surface protein (pIII), cloned into a phagemid vector, expressed in E. coli, and then infected with an M13 helper phage to produce an antibody library in which antibody fragments (scFv or Fab) having sequences of various combinations of heavy and light chain variable regions are displayed on the surface of the phage. From this library, fragments of antibodies that bind to a specific antigen are isolated using a panning method, and after characterizing the isolated antibody fragments, they are converted into whole IgG and expressed in large quantities in animal cells to produce a specific human monoclonal antibody.

[0074] In this specification, the term "phagemid vector" refers to plasmid DNA having a phage origin of replication and generally has an antibiotic resistance gene as a selection marker. In the case of a phagemid vector used for phage display, it contains the gIII gene of M13 phage or a part thereof, and the scFv gene is ligated to the 5' end of the gIII gene and expressed through the transformant.

[0075] In this specification, the term "helper phage" refers to a phage that provides the necessary genetic information for a phagemid to be assembled into a phage particle. Since the phagemid contains only gIII or a portion thereof of the phage gene, the remaining phage gene is supplied by infecting a host cell (transformer) transformed with the phagemid with the helper phage. There are types such as M13K07 or VCSM13, most of which contain antibiotic resistance genes, such as kanamycin, to enable the selection of the transformer infected with the helper phage. Additionally, because the packaging signal is defective, it helps the phagemid gene to be preferentially assembled into the phage particle over the helper phage gene.

[0076] The term “subject” as used in the present invention may be a mammal. Mammals according to the present invention include, but are not limited to, humans, mice, canines, felines, bovines, caprines, equines, ovines, porcines, rodents, lagomorphs, primates, or mammals in utero. The subject may be of both sexes and may be any stage of development. The subject may also be referred to as an “individual or subject(body).”

[0077] In this specification, the term “kit” refers to a packaged product comprising the anti-APP antibody or its antigen-binding fragment or a composition containing the same. The kit preferably comprises a container or box that holds the components of the kit. The box or container may be attached with a protocol or label approved by a pharmaceutical regulatory authority. The components of the present invention are held in a plastic, polyethylene, polypropylene, ethylene, or propylene box or container. The container may be a tube or bottle with a lid. The kit may also include instructions for administering the anti-APP antibody or its antigen-binding fragment or a composition containing the same.

[0078]

[0079] anti-APP antibody or its antigen-binding fragment

[0080] In one aspect of the present invention, the present invention comprises a heavy chain CDR 1 having the amino acid sequence of SEQ ID NO. 3;

[0081] Heavy chain CDR 2 containing the amino acid sequence of SEQ ID NO. 4;

[0082] Heavy chain CDR 3 containing the amino acid sequence of SEQ ID NO. 5;

[0083] Light chain CDR 1 containing the amino acid sequence of SEQ ID NO. 6;

[0084] Light chain CDR 2 comprising the amino acid sequence of SEQ ID NO. 7; and

[0085] Light chain CDR 3 containing the amino acid sequence of SEQ ID NO. 8;

[0086] This relates to an anti-APP antibody or an antigen-binding fragment thereof comprising

[0087] In one aspect of the present invention, the present invention relates to an anti-APP antibody or an antigen-binding fragment thereof comprising a heavy chain variable region of SEQ ID NO. 1 and a light chain variable region of SEQ ID NO. 2.

[0088] In this specification, an anti-APP antibody or its antigen-binding fragment may be named using the above symbols. For example, an anti-APP antibody or its antigen-binding fragment comprising a heavy chain variable region and a light chain variable region comprising the amino acid sequences of SEQ ID NOs 1 and 2, respectively, may be collectively referred to as "M001". An antibody produced using this may be referred to as "M001A" or "M001A-IgG antibody".

[0089] In another aspect of the present invention, the anti-APP antibody or its antigen-binding fragment may specifically bind to APP. Specifically, the anti-APP antibody or its antigen-binding fragment may specifically bind to human or mouse APP.

[0090] In another aspect of the present invention, the anti-APP antibody or its antigen-binding fragment may be single-specific and may specifically bind to a single epitope, namely APP.

[0091] In another aspect of the present invention, the multiple specific antibody molecules may be produced using standard molecular biological techniques known to those skilled in the art (e.g., recombinant DNA and protein expression techniques).

[0092] In another aspect of the present invention, the anti-APP antibody or its antigen-binding fragment is 1 x 10 -8 Equilibrium dissociation constant less than or equal to M (K D It may specifically bind to APP. For example, the anti-APP antibody or its antigen-binding fragment is 5 x 10 -8 Equilibrium dissociation constant less than or equal to M (K D As, specifically, 1×10 -8 M or less, more specifically, 5×10 -9 K less than or equal to M DIt may be specifically bound to a human or mouse APP (e.g., a human or mouse APP).

[0093] In another aspect of the present invention, the equilibrium dissociation constant (K D ) may be a value obtained through surface plasmon resonance (SPR).

[0094] In another aspect of the present invention, the anti-APP antibody or its antigen-binding fragment is 100 nM or less EC 50 It can bind to APP. Specifically, the anti-APP antibody or its antigen-binding fragment may have an EC of 90 nM or less, 80 nM or less, 70 nM or less, 60 nM or less, 50 nM or less, 40 nM or less, 30 nM or less, 20 nM or less, 10 nM or less, 9 nM or less, 8 nM or less, 7 nM or less, 6 nM or less, 5 nM or less, 4 nM or less, 3 nM or less, 2 nM or less, 1 nM or less, 0.9 nM or less, 0.8 nM or less, 0.7 nM or less, 0.6 nM or less, 0.5 nM or less, 0.4 nM or less, 0.3 nM or less, 0.2 nM or less, 0.1 nM or less, or 0.05 nM or less. 50 It can be combined with the APP.

[0095] In another aspect of the present invention, the anti-APP antibody or its antigen-binding fragment is Fab, Fab', Fab'-SH, Fv, single-strand antibody scFv, F(ab')2 fragment, VL, VH, diabody, triabody, tetrabody, minibody ((scFV-CH3)2), IgG-delta CH2, scFv-Fc, (scFv)2-Fc, Fynomer, FynomAbs (fynomers fused to antibodies), dual-affinity re-targeting (DART), AlbudAbs, BiTEs (bispecific T-cell engager), TandAbs (tandem diabodies), DAFs (dual acting Fab), two-in-one antibodies, SMIPs (small modular immunopharmaceuticals), anticalins, FN3 monobody, DARPins, Affibodies, Affilins, Affimers, Affitins, Alphabodies, Avimers, Im7, VLR, VNAR, Trimab, CrossMab, TRIDENT, nanobodies, binanobodies, or D-sdFv, DVD-Igs (dual variable domain immunoglobulin), CovX-bodies (peptide modified antibodies), duobodies, or triomAbs may be, but are not limited thereto.

[0096] In another aspect of the present invention, the anti-APP antibody or its antigen-binding fragment may be a chimeric antibody, a humanized antibody, or a human antibody, but is not limited thereto.

[0097] The anti-APP antibody or its antigen-binding fragment of the present invention can be usefully used to analyze or detect the expression of APP in the cell membrane. For example, by measuring the amount of APP expression in the cell membrane using the anti-APP antibody or its antigen-binding fragment, it can be utilized to diagnose APP-related diseases.

[0098]

[0099] Method for preparing an antibody or its antigen-binding fragment

[0100] In the present invention, the anti-APP antibody or its antigen-binding fragment may be produced by a known method. Specifically, the anti-APP antibody or its antigen-binding fragment may be produced using antibody display technology, for example, phage library technology.

[0101] In one embodiment of the present invention, the anti-APP antibody or a monoclonal antibody, which is one of its antigen-binding fragments, can be produced by injecting an APP antigen into a test subject, e.g., a mouse, by a known method, and then isolating hybridoma cells that express an antibody containing a desired sequence or functional feature.

[0102] In another embodiment of the present invention, a DNA fragment encoding a monoclonal antibody can be isolated and sequenced using a conventional method (e.g., using an oligonucleotide probe capable of specifically binding to genes encoding the heavy and light chains of the monoclonal antibody). Specifically, the isolated DNA fragment can be placed into a recombinant expression vector and transfected into a host cell, e.g., E. coli cell, Simian COS cell, CHO cell, or myeloma cell that does not otherwise produce immunoglobulin protein, to obtain the synthesis of a monoclonal antibody in the recombinant host cell.

[0103]

[0104] Polynucleotides, Vectors, and Host Cells

[0105] In one aspect of the present invention, the invention relates to a polynucleotide encoding the anti-APP antibody or the antigen-binding fragment thereof.

[0106] In another aspect of the invention, the polynucleotide may be present in whole cells or cell lysates, or may be present in a purified or substantially pure form. Specifically, the polynucleotide may be a “separated” or “substantially purely isolated” nucleic acid when other cell components or other contaminants, such as other cell nucleic acids or proteins, are purified and removed by standard techniques, e.g., alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, or other methods widely known in the art.

[0107] In another aspect of the present invention, the polynucleotide may be, for example, DNA or RNA, and may or may not include an intron sequence. Specifically, the polynucleotide may be a cDNA molecule.

[0108] In one embodiment of the present invention, the polynucleotide may comprise a polynucleotide encoding a heavy chain region, a light chain region, or both a heavy chain and a light chain region of the anti-APP antibody or its antigen-binding fragment, and specifically, may comprise a polynucleotide encoding a heavy chain variable region, a light chain variable region, or both a heavy chain and a light chain variable region.

[0109] In another embodiment of the present invention, the polynucleotide may include a polynucleotide that encodes / encodes the heavy chain variable region of SEQ ID NO. 1 or encodes the light chain variable region of SEQ ID NO. 2.

[0110] In another embodiment of the present invention, the VL and / or VH-coding DNA fragment may be further manipulated, for example, by standard recombinant DNA technology to produce a variable region gene into a full-length antibody chain, a Fab fragment, or an antibody in the form of a scFv. Additionally, the VL- or VH-coding DNA fragment may be operably coupled to other DNA fragments encoding antibody constant regions, such as the hIgG1 Fc region (hFc), the hIgG4 Fc region (e.g., hIgG4 (S228P) with the S228P mutation), the hCκ region, or a flexible linker.

[0111] In another embodiment of the present invention, the VH-encoding DNA fragment can be produced into a full-length heavy chain gene by operably coupling it to another DNA molecule encoding a heavy chain constant region (CH1, CH2, or CH3). Specifically, the heavy chain constant region may be the heavy chain constant region of IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD. More specifically, the heavy chain constant region may be the heavy chain constant region of IgG4, and even more specifically, the heavy chain constant region of hIgG4 (S228P).

[0112] In another embodiment of the present invention, in the heavy chain gene of the Fab fragment, the VH-coding DNA fragment can be operably coupled to another DNA molecule encoding the heavy chain CH1 constant region.

[0113] In another embodiment of the present invention, in the scFv gene, the VL- and / or VH-coding DNA fragment may be operably coupled to a flexible linker, for example, another fragment encoding the amino acid sequence (Gly4-Ser)3. Additionally, the VL- and / or VH-coding DNA fragment may be expressed as a continuous single-chain protein comprising VL- and / or VH- sites coupled by the flexible linker.

[0114] In one aspect of the present invention, the present invention relates to a recombinant expression vector comprising the polynucleotide.

[0115] In one embodiment of the present invention, the polynucleotide may be isolated from various sources and genetically engineered and / or amplified and / or expressed by recombination. Specifically, it may be expressed by any recombinant expression system including bacteria, yeast, insects, or mammals.

[0116] In another embodiment of the present invention, the method of manipulating the polynucleotide includes, but is not limited to, subcloning into an expression vector, labeling probe, sequencing, or hybridization. The method of manipulating the polynucleotide may be performed as known in the art.

[0117] In another aspect of the present invention, the invention relates to a host cell comprising the recombinant expression vector.

[0118] In one embodiment of the present invention, the host cell may include, but is not limited to, a human cell, an animal cell, a plant cell, an insect cell, a fungal cell, or a bacterial cell.

[0119] In another embodiment of the present invention, the human cell may include, but is not limited to, HeLa cells, 911 cells, AT1080 cells, A549 cells, A293 cells, HEK293 cells, or Expi293F cells.

[0120] In another embodiment of the present invention, the animal cell may include Chinese hamster ovary cell (CHO cell), Simian COS cell, BHK cell, NSO cell, or Bowes melanoma cell, but is not limited thereto.

[0121] In another embodiment of the present invention, the plant cell may include Nicotiana tabacum, Arabidopsis thaliana, Zea mays, Glycine max, or Solanum tuberosum cells, but is not limited thereto.

[0122] In another embodiment of the present invention, the insect cell may include Drosophila melanogaster cells or SF9 (Spodoptera frugiperda) cells, but is not limited thereto.

[0123] In another embodiment of the present invention, the fungal cell may include yeast cells, for example, Pichia pastoris, Saccharomyces cerevisiae, or Hansenula polymorpha, but is not limited thereto.

[0124] In another embodiment of the present invention, the bacterial cells may include gram-positive bacterial cells, gram-negative bacterial cells, bacterial cells of the genus Escherichia or bacterial cells of the genus Pseudomonas, but are not limited thereto.

[0125]

[0126] Composition, Use, Method, and Kit

[0127] In one aspect of the present invention, the present invention relates to a method for detecting the presence of APP in a sample using the anti-APP antibody or its antigen-binding fragment. Specifically, the present invention relates to a method for measuring the amount of APP expression in a sample using the anti-APP antibody or its antigen-binding fragment.

[0128] In one aspect of the present invention, a method for detecting the presence of APP in a sample or a method for measuring the amount of APP expression in a sample may include the step of contacting the anti-APP antibody or its antigen-binding fragment with a sample and a control sample under conditions in which the anti-APP antibody or its antigen-binding fragment can bind to APP to form a complex.

[0129] In one embodiment of the present invention, the sample is obtained from an individual or object and may be, for example, cells, blood, plasma, serum, bone marrow fluid, lymph fluid, saliva, tear fluid, mucosal fluid, amniotic fluid, or a combination thereof.

[0130] In another aspect of the present invention, the present invention relates to a composition for analyzing or detecting cell membrane APP expression, comprising the anti-APP antibody or an antigen-binding fragment thereof.

[0131] In another aspect of the present invention, the present invention relates to a composition for diagnosing APP-related diseases comprising the anti-APP antibody or an antigen-binding fragment thereof.

[0132] The above APP-related diseases may be cancer, Alzheimer's disease, Creutzfeldt-Jakob disease, multiple sclerosis, Parkinson's disease, Down syndrome, amyloidosis, or polyneuropathy, and are not limited to diseases that can be diagnosed by comparing the presence or level of APP with a control group.

[0133] In another aspect of the present invention, the invention relates to the use of the anti-APP antibody or its antigen-binding fragment for analyzing or detecting the expression of cell membrane APP in cells isolated from a cultured cell line or individual.

[0134] In another aspect of the present invention, the invention relates to the use of the anti-APP antibody or its antigen-binding fragment for diagnosing APP-related diseases.

[0135] The above APP-related diseases may be cancer, Alzheimer's disease, Creutzfeldt-Jakob disease, multiple sclerosis, Parkinson's disease, Down syndrome, amyloidosis, or polyneuropathy.

[0136] In another aspect of the present invention, the present invention may be a method for providing information on APP-related diseases, comprising the step of measuring the cell membrane APP expression level in cells isolated from a cultured cell line or individual using the anti-APP antibody or its antigen-binding fragment.

[0137] In one embodiment of the present invention, the step of measuring the APP expression level may include measuring by electrophoresis, immunoblotting, enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction, Northern blotting, polymerase chain reaction (PCR), real-time polymerase chain reaction (or quantitative PCR: QPCR), reverse transcription polymerase chain reaction (rt PCR) protein chip, immunoprecipitation, microarray, electron microscopy, or a combination thereof. The electrophoresis may be SDS-PAGE, isoelectric point electrophoresis, two-dimensional electrophoresis, or a combination thereof. The PCR may be real-time PCR or reverse transcription PCR.

[0138] In another aspect of the present invention, the method for providing APP-related disease information may include a method for diagnosing APP-related diseases.

[0139] The method for providing information on APP-related diseases or the method for diagnosing APP-related diseases may include a step of comparing the measured APP expression level with a normal control group.

[0140] In another aspect of the present invention, the present invention relates to a kit for diagnosing APP-related diseases comprising the anti-APP antibody or its antigen-binding fragment or a diagnostic composition containing the same.

[0141] In one embodiment of the present invention, the kit may additionally include a user manual describing optimal reaction performance conditions. The manual may include a pamphlet, a guide booklet in the form of a leaflet, a label attached to the kit, or a description on the surface of a package containing the kit. Additionally, the manual may include information disclosed or provided through an electronic medium, such as the Internet.

[0142] In one aspect of the present invention, the present invention may be in a form in which the anti-APP antibody or a fragment thereof is conjugated with a detectable label.

[0143] In another aspect of the present invention, the aforementioned anti-APP antibody or a fragment thereof may be combined with a fluorescent substance intended for detection through a linker to be prepared as an antibody-fluorescent conjugate.

[0144] In one embodiment of the present invention, the anti-APP antibody or its antigen-binding fragment may be labeled with a detectable label directly or indirectly. For example, the anti-APP antibody or its antigen-binding fragment may be labeled by directly labeling it with a fluorescent substance. An indirect labeling method may involve using a secondary antibody containing a detectable label, wherein the secondary antibody binds to the anti-APP antibody or its antigen-binding fragment. Another indirect labeling method may involve biotinylating the anti-APP antibody or its antigen-binding fragment, wherein the biotinylated anti-APP antibody or its antigen-binding fragment may be detected using streptavidin or avidin containing a detectable label.

[0145] The present invention will be described in more detail below through examples. These examples are intended solely to explain the invention more specifically, and it will be obvious to those skilled in the art that the scope of the invention is not limited by these examples according to the gist of the invention.

[0146] Example 1: Preparation of Human APP-Specific Binding Antibody

[0147] Example 1-1. Mouse Immunization Response and Development of Anti-APP Antibody via Hybridoma Cells

[0148] Recombinant human APP (Amyloid-beta precursor protein) was injected subcutaneously into four BALB / c mice mixed with an adjuvant (Sigma) as an antigen. Subsequently, blood samples were collected from the mice, and antibody production was confirmed via ELISA. After two immunizations, spleens were removed from the immunized mice, B lymphocytes were isolated, and then fused with cultured myeloma cells (sp2 / 0). The fused cells were cultured in a medium (HAT medium) supplemented with hypoxanthin, aminopterine, and thymidine to selectively select and culture hybridomas (myeloma-B lymphocyte fusion cells). Among the obtained hybridoma cells, those producing antibodies that reacted with the antigen were identified via ELISA. Subsequently, five types of monoclonal hybridoma cells were produced using a cloning method based on serial dilution.

[0149] Using the final culture medium of five types of monoclonal hybridoma cell clones, binding to human APP antigens via ELISA and binding to HEK293FT cells temporarily expressing human APP via FACS were evaluated to select a final clone. The sequences of the heavy and light chain variable regions of the selected clone were obtained through RNA sequencing analysis. The obtained variable region sequences of the antibodies are shown in Table 1 below, and furthermore, the CDR sequences of the antibodies are shown in Table 2. The CDR regions were designated based on IMGT rules.

[0150] Heavy chain variable region (VH) Sequence No. 1 EVQLEESGGDLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLEWVATISSGGFYTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCASLLRLPSWFVYWGQGTLVTVSA Light chain variable region (VL) Sequence No. 2 DIQLTQCPASLSASVGETVTITCRASGNIHNYLAWYQQKQGKSPQLLVYNAKTLADGVPSRFSGSGSGTQYSLKINSLQPEDFGSYYCQHFWSTPWTFGGGTKLEIK

[0151] CDR1CDR2CDR3VH Sequence No. 3GFTFSSY Sequence No. 4SSGGFY Sequence No. 5LLRLPSWFVYVL Sequence No. 6RASGNIHNYLA Sequence No. 7NAKTLAD Sequence No. 8QHFWSTPWT

[0152] Example 1-2. Preparation of Anti-APP Antibody

[0153] An anti-APP antibody was prepared using the amino acid sequences of the heavy chain and light chain variable domains obtained in Example 1-1. At this time, the heavy chain variable domain was connected to the IgG4 heavy chain constant domain into which the S228P mutation was introduced (Sequence No. 9), and the light chain variable domain was connected to the light chain constant domain of human kappa (Sequence No. 10) (Table 3).

[0154] sequence number 9ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVH NAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKSEQ ID NO. 10RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0155] After modifying the heavy and light chain sequence information of the antibody into DNA base sequences through codon optimization, a temporary expression vector was constructed by introducing it into the pcDNA3.4 expression vector. Subsequently, transient transfection was performed on the ExpiCHO-S cell line, and antibody expression was carried out by culturing for 8 days.

[0156] The expression antibody was purified using affinity chromatography with Protein A. Specifically, the antibody was conjugated to an affinity chromatography resin equilibrated with TRIS buffer (pH 7.5), and the target protein was eluted with 100 mM glycine buffer (pH 3.4). Subsequently, the protein solution was titrated to pH 5 with 1 M sodium acetate, formulated with 50 mM acetate buffer (pH 5.0) containing 150 mM sodium chloride, and concentrated using a 50 kDa cut-off Amicon filter to complete the preparation of the final antibody. The prepared antibody was named M001A, its concentration was measured using a Nanodrop 2000, and its purity was confirmed by analysis using SDS-PAGE and SEC-HPLC. The SDS-PAGE results of the produced material are shown in Fig. 1 and the SEC-HPLC results are shown in Fig. 2, respectively, and the results of the measured concentration and SEC-HPLC purity are as shown in Table 4 below.

[0157]

[0158] Substance Concentration (g / L) Purity (SEC-HPLC, %) M001A12.5396.2

[0159]

[0160] Examples 1-3. Preparation of Anti-APP Antibody-Fluorescent Conjugate

[0161] An anti-APP antibody-fluorescent conjugate was prepared by conjugating a fluorescent substance to the M001A antibody prepared above. The fluorescent substance used was Alexa Fluor™ 647 NHS Ester (Thermo Fisher).

[0162] Specifically, the above M001A was titrated with a 7.5% chlorocarbonate solution to a pH range of 7–8, and the fluorescent substance was added at a ratio of 30 moles per mole of antibody. At this time, the fluorescent substance was dissolved in DMSO to a concentration of 2 mg / mL prior to addition. The final mixture was reacted for 1 hour under laboratory room temperature conditions. After the reaction was completed, the mixture was loaded onto a Desalting column (HiTrap Desalting column with Sephadex G-25, Cytiva) equilibrated with 1X PBS buffer to remove unreacted fluorescent substance.

[0163] Subsequently, the final antibody-fluorescent conjugate was prepared by concentrating using a 50 kDa cut-off Amicon filter. The prepared anti-APP antibody-fluorescent conjugate was named M001A-AF647, and its concentration was measured using a Nanodrop 2000 and its purity was confirmed by SEC-HPLC analysis. The SEC-HPLC results are shown in Figure 3.

[0164] Additionally, the above anti-APP antibody-fluorescent conjugate (M001A-AF647) was diluted to 0.5 mg / mL using Nanodrop 2000, and the absorbance at wavelengths of 280 nm and 650 nm was checked. Using this, the number of fluorescent substances bound per antibody was determined. The measured concentrations, SEC-HPLC purity, and the results regarding the number of fluorescent substances bound are shown in Table 5 below.

[0165] Substance Concentration (g / L) Purity (SEC-HPLC, %) Number of Fluorescent Substances Bound (Molar Ratio) M001A-AF647 1.839 0.67.3

[0166] Example 2: Human APP binding ability of anti-APP antibody

[0167] Example 2-1. APP binding ability of anti-APP antibody (ELISA analysis)

[0168] An ELISA (Enzyme-Linked Immunosorbent Assay) was performed to confirm the ability of the antibody M001A prepared in Examples 1-2 above to bind to human APP.

[0169] A human APP solution at a concentration of 25 nM was prepared by diluting the antigen protein, human APP (GenScript, China), in PBS buffer. 50 μL of the prepared human APP solution was added to each well of a 96-well plate (Costar, USA, Cat. No. 3690) and coated overnight at 4°C. The next day, all solutions in the 96-well plate were removed, and blocking buffer (3% skim milk in PBS) was added to each well, followed by incubation at room temperature for 1 hour. M001A antibody and isotype control mouse IgG1 (mIgG1) antibody samples were sequentially diluted with blocking buffer at 10-fold dilution ratios from 200 nM to 8 points (0.02 pM). After the blocking process was complete, the buffer was removed from each well, and 50 μL of the diluted antibody was added to each well and incubated at room temperature for 1 hour. The wells were washed with PBST (0.1% Tween 20 in PBS), and for antibody detection, the secondary antibody, HRP-conjugated anti-mouse IgG Fc antibody (Jackson Immuno Research, USA, Cat. No.: 115-035-164) (1:100,000), was added at room temperature for 1 hour, followed by washing again with PBST. For color development, 50 μL of TMB solution (Thermo Fisher Scientific, USA, Cat. No.: 34028) was added to each well and incubated at room temperature for 10 minutes, after which the absorbance at 450 nm (optical density at 450 nm, OD) 450) was measured. OD according to antibody treatment concentration 450 The values ​​are shown in Fig. 4.

[0170] EC to indicate the binding affinity level of the M001A antibody to human APP 50 The concentration (half maximal effective concentration) was calculated (Table 6). EC of M001A antibody 50 (nM) was 0.193, confirming that it has excellent binding ability to human APP.

[0171]

[0172] Test articleEC 50 (nM)M001A0.193

[0173] Example 2-2. APP binding ability of anti-APP antibody (FACS test)

[0174] The ability of antibody M001A prepared in Examples 1-2 above to bind to human APP was analyzed by FACS (Fluorescence Activated Cell Sorting, Flow Cytometry) (FACS Canto™ II, BD, USA).

[0175] HEK293 cells (Thermo Fisher Scientific, USA) were transfected using a plasmid vector containing nucleotides encoding APP, and HEK293 cells expressing human APP antigens were named HEK293 / hAPP cells.

[0176] 1 x 10⁶ suspended in FACS buffer 6HEK293 / hAPP cells at a concentration of 100 μL were seeded into 96-well plates. Additionally, M001A antibody and human IgG4 antibody samples, which served as an isotype control, were sequentially diluted in FACS buffer at 5-fold dilution ratios starting from 30 μg / mL to 10 points (0.02 ng / mL). The diluted antibodies were added to the 96-well plates seeded with cells and incubated at 4°C for 1 hour. Each well was washed with FACS buffer, Goat Anti-Human IgG(H+L) Cross-Adsorbed Secondary Antibody (Alexa Fluor™ 647 (Thermo Fisher Scientific, USA, Cat. No.: A21445) (1:400)), and the 96-well plates were further incubated before being washed with FACS buffer. The degree of antigen-antibody binding was measured using a flow cytometer as the gMFI (Geometric Mean Fluorescence Intensity) value of allophycocyanin (APC) (Table 7), and the gMFI values ​​according to antibody treatment concentration are shown in Figure 5.

[0177] EC to indicate the degree of binding affinity of the M001A antibody to antigen proteins expressed on the cell surface 50 The concentration was calculated (Table 7). EC of M001A antibody 50 (nM) was 9.88, confirming that the antibody has excellent binding ability to human APP expressed on the cell surface.

[0178]

[0179] Test articleEC 50 (nM)M001A9.88

[0180] Example 3: Evaluation of the inhibitory effect of anti-APP antibodies on the interaction between human APP and human CNTN4 (Competitive ELISA assay)

[0181] A competitive ELISA (Competitive Enzyme-Linked Immunosorbent Assay) test was performed to confirm the ability of antibody M001A prepared in Examples 1-2 above to inhibit the interaction between human APP and human CNTN4 proteins.

[0182] Human APP (GenScript, China), an antigen protein, was diluted in PBS buffer to prepare a human APP solution at a concentration of 10 nM. 50 μL of the prepared human APP solution was added to each well of a 96-well plate (Costar, USA, Cat. No. 3690) and coated overnight at 4°C. The next day, all solutions in the 96-well plate were removed, and blocking buffer (3% skim milk in PBS) was added to each well, followed by incubation at room temperature for 1 hour. M001A antibody and mouse IgG1 (mIgG1) antibody samples, an isotype control, were diluted in blocking buffer and prepared sequentially at 8 points (0.01 nM) starting from 800 nM, which is twice the final concentration, at a dilution ratio of 5 times. The competing protein, human CNTN4-RbFc (BioIntron, China), was diluted in blocking buffer to prepare a human CNTN4-RbFc protein solution at a concentration of 20 nM, which is twice the final concentration. A mixture was prepared by mixing the prepared antibody solution at twice the concentration and the human CNTN4-RbFc protein solution in a 1:1 ratio. After the blocking process was completed, the buffer was removed from each well, and 50 μL of the prepared mixture was added to each well and incubated at room temperature for 1 hour. The wells were washed with PBST (0.1% Tween 20 in PBS), and for the detection of the competing protein, the secondary antibody, HRP-conjugated anti-mouse IgG Fc antibody (Jackson Immuno Research, USA, Cat. No.: 115-035-164) (1:10,000), was treated at room temperature for 1 hour, followed by washing again with PBST. For color development, TMB solution (TMB solution, Thermo Fisher Scientific, USA, Cat. No.50 μL of : 34028) was added to each well and reacted at room temperature for 10 minutes, then the absorbance at 450 nm (optical density at 450 nm, OD. 450 ) was measured. The degree of APP / CNTN4 binding (APP / CNTN4 binding, %) according to antibody treatment concentration is shown in Fig. 6.

[0183] To evaluate the level of inhibitory activity of the M001A antibody on the interaction between human APP and human CNTN4 protein, IC 50 The concentration (half maximal inhibitory concentration) was calculated (Table 8). The IC50 of the M001A antibody 50 (nM) was 7.87, confirming that it has excellent inhibitory activity against APP / CNTN4 interactions.

[0184]

[0185] Test articleIC 50 (nM)M001A7.87

[0186] Example 4: Evaluation of the specificity of anti-APP antibody (Cell microarray analysis)

[0187] An off-target screening (Charles River, UK) test was conducted to confirm whether the antibody M001A prepared in Examples 1-2 above specifically binds to APP.

[0188] The interactions between M001A and each protein were screened using a cell microarray system on a library of 6,019 human plasma membrane proteins (Charles River) (Table 9). Specific interactions of the M001A antibody with the primary target APP were confirmed, and no additional binding to other proteins was found. Therefore, it was confirmed that M001A has specific binding to APP without binding to any other proteins.

[0189]

[0190] Gene IDAPPAPPAPPUniProt IDP05067-8P05067-1P05067-10Unique clone number (UCN)148017113566ClassificationPM * PMPMRef seq information(for specific hits)Isoform APP751Isoform APP770Isoform APP639Test articleM001A(0.25 mg / mL)Weak / Med 1 Weak 2 -Mouse IgG1(2 mg / mL)--PBS(2 nd antibody only)---

[0191] * PM: plasma membrane

[0192] 1 Weak / Med: specific hits of weak / medium intensity or above (not seen with negative control molecule or previous PBS only control)

[0193] 2Weak: specific hits of weak intensity (not seen with negative control molecule or previous PBS only control)

[0194]

[0195] Example 5: Confirmation of App expression using M001A-AF647 in App wild-type and knockout mouse T cells

[0196] Western blotting and flow cytometry tests were performed to confirm the specificity of APP expression on mouse T cell membranes using the antibody of the present invention in App wild-type and knockout mouse T cells.

[0197] Spleens were removed from 10-week-old App wild-type and knockout mice and unicellularized to produce splenocytes. One-quarter of these were lysed to measure protein concentration, and a total of 20 μg of protein was transferred to an SDS-PAGE (sodium dodecyl sulfate-polyacrylamide) gel for electrophoresis. The proteins separated by electrophoresis were transferred to a filter membrane, and Western blotting was performed using anti-APP and anti-GAPDH antibodies to plot the chemiluminescence reaction results (Fig. 7a). As a result, App was detected only in the splenocytes of App wild-type mice and not in knockout mice, confirming that this is a mouse model capable of verifying the expression specificity of App.

[0198] CD4 from the remaining 3 / 4 of the splenocytes + and CD8 +T cells were isolated, and clonal expansion T cells were prepared by treating them with mouse anti-CD3 / 28 antibodies (2 μg / mL each) and recombinant IL-2 protein (100 U / mL). Activated T cells were produced by coating clonal expansion T cells with mouse anti-CD3 antibody (2 μg / mL) diluted in PBS in a cell culture dish and culturing them for 16 hours. Flow cytometry was performed on clonal expansion T cells with and without activation to detect fluorescence, and the results were plotted (Fig. 7b). As a result, the anti-APP antibody-fluorescent conjugate (M001A-AF647) prepared in Example 1-3 was applied to activated App wild-type mouse CD4 + and CD8 + Increased expression of App could be detected only in T cell membranes, and was not detected in knockout mice.

[0199]

[0200] Example 6: Confirmation of APP expression in human T cells using M001A-AF647

[0201] Reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry tests were performed to confirm APP expression in human T cells using the antibody of the present invention.

[0202] CD4 from human peripheral blood mononuclear cells + and CD8 + T cells were isolated and clonal expansion T cells for co-culture were prepared by treating them with human anti-CD3 / 28 antibody (2 μg / mL each) and IL-2 recombinant protein (100 U / mL). Human anti-CD3 antibody (2 μg / mL) diluted in PBS was coated onto a cell culture dish for 16 hours, and the clonal expansion T cells were cultured for 16 hours to produce activated T cells.

[0203] Clonal expansion T cells with and without activation were lysed with Trizol to extract RNA, and the concentration was measured. A total of 2 μg of RNA was used to synthesize cDNA using reverse transcriptase. PCR was performed using the cDNA and APP and GAPDH-specific primers, and the results of the electrophoresis of the PCR-amplified APP and GAPDH were plotted on an agarose gel (Fig. 8a). As a result, activated human CD4 + and CD8 + Increased expression of APP in T cells was confirmed.

[0204] The results of fluorescence detection were plotted by flow cytometry after treating clonal expansion T cells with and without activation with the anti-APP antibody-fluorescent conjugate (M001A-AF647, 3 μg / mL) prepared in Examples 1-3 above (Fig. 8b). As a result, the anti-APP antibody-fluorescent conjugate (M001A-AF647) prepared in Examples 1-3 above [indicated] activated human CD4 + and CD8 + Increased expression of APP in the T cell membrane could be detected.

Claims

1. Heavy chain CDR 1 containing the amino acid sequence of SEQ ID NO. 3; Heavy chain CDR 2 containing the amino acid sequence of SEQ ID NO. 4; Heavy chain CDR 3 containing the amino acid sequence of SEQ ID NO. 5; Light chain CDR 1 containing the amino acid sequence of SEQ ID NO. 6; Light chain CDR 2 comprising the amino acid sequence of SEQ ID NO. 7; and Light chain CDR 3 containing the amino acid sequence of SEQ ID NO. 8; An anti-APP (Amyloid-beta precursor protein) antibody or an antigen-binding fragment thereof comprising 2. An anti-APP antibody or an antigen-binding fragment thereof comprising the heavy chain variable region of SEQ ID NO. 1 and the light chain variable region of SEQ ID NO.

2.

3. In Paragraph 1 or 2, 1 X 10 -8 Equilibrium dissociation constant less than or equal to M (K D An anti-APP antibody or its antigen-binding fragment that binds to APP.

4. In Paragraph 1 or 2, EC of 100 nM or less 50 An anti-APP antibody or its antigen-binding fragment that binds to APP.

5. In claim 1 or 2, Fab, Fab', Fab'-SH, Fv, single-strand antibody scFv, F(ab')2 fragment, VL, VH, diabody, triabody, tetrabody, minibody((scFV-CH3)2), IgG-delta CH2, scFv-Fc, (scFv)2-Fc, Fynomer, FynomAbs (fynomers fused to antibodies), dual-affinity re-targeting (DART), AlbudAbs, BiTEs (bispecific T-cell engager), TandAbs (tandem diabodies), DAFs (dual acting Fab), two-in-one antibodies, SMIPs (small modular immunopharmaceuticals), anticalins, FN3 monobody, DARPins, Affibodies, An anti-APP antibody or its antigen-binding fragment that is Affilins, Affimers, Affitins, Alphabodies, Avimers, Im7, VLR, VNAR, Trimab, CrossMab, TRIDENT, a nanobody, binanobodies, or D-sdFv, DVD-Igs (dual variable domain immunoglobulin), CovX-bodies (peptide modified antibodies), duobodies, or triomAbs.

6. An anti-APP antibody or an antigen-binding fragment thereof, which is a chimeric antibody, a humanized antibody, or a human antibody, according to claim 1 or 2.

7. An antibody conjugate comprising the anti-APP antibody of claim 1 or 2 or an antigen-binding fragment thereof and a detectable label.

8. A polynucleotide encoding the anti-APP antibody of claim 1 or 2 or its antigen-binding fragment.

9. A recombinant expression vector comprising the polynucleotide of claim 8.

10. A composition for analyzing or detecting the expression of a cell membrane APP, comprising the anti-APP antibody of claim 1 or 2 or an antigen-binding fragment thereof.

11. A composition for diagnosing APP-related diseases comprising the anti-APP antibody of claim 1 or 2 or an antigen-binding fragment thereof.

12. A composition according to claim 11, wherein the APP-related disease is cancer, Alzheimer's disease, Creutzfeldt-Jakob disease, multiple sclerosis, Parkinson's disease, Down syndrome, amyloidosis, or polyneuropathy.

13. A method for providing APP-related disease information, comprising the step of measuring the cell membrane APP expression level in cells isolated from a cultured cell line or individual using the anti-APP antibody of claim 1 or 2 or an antigen-binding fragment thereof.

14. In claim 13, the APP-related disease is cancer, Alzheimer's disease, Creutzfeldt-Jakob disease, multiple sclerosis, Parkinson's disease, Down syndrome, amyloidosis, or polyneuropathy.

15. A kit for diagnosing APP-related diseases comprising the composition of Claim 11 above.

16. Use of the anti-APP antibody of claim 1 or 2 or its antigen-binding fragment for analyzing or detecting cell membrane APP expression in cells isolated from a cultured cell line or individual.

17. Use of the anti-APP antibody of claim 1 or 2 or its antigen-binding fragment for diagnosing APP-related diseases.

18. In claim 17, the APP-related disease is cancer, Alzheimer's disease, Creutzfeldt-Jakob disease, multiple sclerosis, Parkinson's disease, Down syndrome, amyloidosis, or polyneuropathy.

Images