Antibody and application thereof

Abstract

An object of the present disclosure is to provide a novel method for detecting differentiated thyroid cancer.SOLUTION: In one aspect, the present disclosure provides the following invention. An antibody or binding fragment thereof capable of binding to a peptide represented by the amino acid sequence ANHHKAHH (SEQ ID NO: 1).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to antibodies and applications thereof. Specifically, the present disclosure relates to anti-FAM19A2 antibodies and applications thereof. [Background technology]

[0002] Thyroid cancer is a malignant tumor that develops in the thyroid gland. Thyroid cancer is classified into differentiated carcinoma (or differentiated thyroid carcinoma), medullary carcinoma, and undifferentiated carcinoma. Papillary carcinoma and follicular carcinoma are known as subtypes of differentiated thyroid carcinoma.

[0003] Papillary carcinoma accounts for approximately 90% of thyroid cancers. It is known that papillary carcinoma generally progresses more slowly than other cancers and is prone to metastasis to lymph nodes.

[0004] On the other hand, follicular carcinoma accounts for about 5% of thyroid cancers. Follicular carcinoma can metastasize to distant organs (e.g., lungs and bones) via the bloodstream (hematogenous metastasis). It is known to be difficult to distinguish from benign tumors in thyroid ultrasound or fine-needle aspiration cytology tests.

[0005] Patent Document 1 discloses a method for detecting follicular cancer by detecting the expression of FAM19A2. Specifically, Patent Document 1 discloses a method for detecting follicular cancer by detecting FAM19A2 mRNA. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO2022 / 210783 issue Summary of the Invention [Problem to be solved by the invention]

[0007] As described above, papillary carcinoma and follicular carcinoma are known subtypes of differentiated thyroid carcinoma, and there is a need for a highly accurate detection method for detecting differentiated thyroid carcinoma. The present disclosure aims to provide a new method for detecting differentiated thyroid carcinoma. [Means for solving the problem]

[0008] In order to achieve the above object, the present disclosure includes, in one aspect, the following invention. (Invention 1) An antibody or a binding fragment thereof, which is capable of binding to a peptide represented by the amino acid sequence ANHHKAHH (SEQ ID NO: 1). (Invention 2) The antibody or binding fragment thereof of invention 1, binds to a truncated fragment of FAM19A2 having the amino acid sequence ANHHKAHH (SEQ ID NO: 1); does not bind to a cleavage fragment of FAM19A2 that does not have the amino acid sequence ANHHKAHH (SEQ ID NO: 1); An antibody or binding fragment thereof. (Invention 3) 3. The antibody or binding fragment thereof according to Invention 1 or 2, which is a monoclonal antibody. (Invention 4) A composition comprising the antibody or binding fragment thereof according to any one of Inventions 1 to 3, Used to detect differentiated thyroid cancer in the thyroid gland, composition. (Invention 5) The composition of invention 4, wherein the differentiated thyroid carcinoma is follicular carcinoma. (Invention 6) The composition of invention 4, wherein the differentiated thyroid carcinoma is papillary carcinoma. (Invention 7) 7. The composition according to any one of Inventions 4 to 6, which is used for detecting differentiated thyroid cancer in the thyroid gland by an immunohistochemical staining technique. (Invention 8) A detection kit comprising the antibody or binding fragment thereof according to any one of Inventions 1 to 3, Used to detect differentiated thyroid cancer in the thyroid gland, Detection kit. (Invention 9) 1. A method for detecting differentiated thyroid cancer in the thyroid gland of a subject, said method being an in vitro method comprising: The method comprises: detecting the presence of FAM19A2 having the amino acid sequence ANHHKAHH (SEQ ID NO: 1) in a biological sample taken outside the body of a subject using the antibody or binding fragment thereof according to any one of Inventions 1 to 3; A method comprising: (Invention 10) 4. The antibody or binding fragment thereof according to any one of Inventions 1 to 3, which is used for detecting, treating, and / or diagnosing differentiated thyroid carcinoma in the thyroid gland. (Invention 11) Use of the antibody or binding fragment thereof according to any one of Inventions 1 to 3 for detecting differentiated thyroid carcinoma in the thyroid gland. (Invention 12) Use of the antibody or binding fragment thereof according to any one of inventions 1 to 3 for producing a composition for detecting, treating, and / or diagnosing differentiated thyroid carcinoma in the thyroid gland. (Invention 13) Use of the antibody or binding fragment thereof according to any one of Inventions 1 to 3 for producing a kit for detecting, treating, and / or diagnosing differentiated thyroid carcinoma in the thyroid gland. (Invention 14) 1. A method for detecting differentiated thyroid cancer of the thyroid gland in a subject having or suspected of having differentiated thyroid cancer of the thyroid gland, the method comprising: a step of contacting a biological sample with the antibody or binding fragment thereof of any one of Inventions 1 to 3, and detecting the presence of FAM19A2 in the biological sample by detecting the binding of FAM19A2 having the amino acid sequence ANHHKAHH (SEQ ID NO: 1) to the antibody or binding fragment thereof; Including, The biological sample is at least a portion of a thyroid gland obtained from the subject. method. (Invention 15) 1. A method of diagnosing differentiated thyroid cancer in a subject, the method comprising: obtaining a biological sample from the subject; contacting the biological sample with the antibody or binding fragment thereof of any one of Inventions 1 to 3, and detecting the presence of FAM19A2 in the biological sample by detecting the binding between FAM19A2 having the amino acid sequence ANHHKAHH (SEQ ID NO: 1) and the antibody or binding fragment thereof; If FAM19A2 is detected, diagnosing the subject with differentiated thyroid cancer; A method comprising: (Invention 16) 1. A method of treating a subject with differentiated thyroid cancer in the thyroid gland, the method comprising: obtaining a biological sample from the subject; contacting the biological sample with the antibody or binding fragment thereof of any one of Inventions 1 to 3, and detecting the presence of FAM19A2 in the biological sample by detecting the binding between FAM19A2 having the amino acid sequence ANHHKAHH (SEQ ID NO: 1) and the antibody or binding fragment thereof; If FAM19A2 is detected, diagnosing the subject with differentiated thyroid cancer; administering to the subject a therapeutically effective amount of a therapeutic agent for differentiated thyroid cancer; A method comprising: (Invention 17) 17. The method of claim 15 or 16, wherein the antibody or binding fragment thereof is a non-human antibody or binding fragment thereof. (Invention 18) 17. The method of claim 15 or 16, wherein the applying step comprises administering a therapeutically effective amount of an antibody or a binding fragment thereof capable of binding to a peptide represented by the amino acid sequence ANHHKAHH (SEQ ID NO: 1). [Effects of the Invention]

[0009] In one aspect, the invention relates to an antibody or a binding fragment thereof capable of binding to the peptide represented by the amino acid sequence ANHHKAHH (SEQ ID NO: 1), which can specifically detect areas characteristic of differentiated thyroid cancer. [Brief explanation of the drawings]

[0010] [Figure 1] The amino acid sequence of the recombinant FAM19A2 antigen, in which enhanced green fluorescent protein and six histidine residues were added to the human FAM19A2 sequence (Q8N3H0), is shown. [Figure 2] The amino acid sequence of FAM19A2 is shown below (sp|Q8N3H0|TAFA2_HUMAN Chemokine-like protein TAFA-2 OS=Homo sapiens OX=9606 GN=TAFA2 PE=2 SV=1). Within the human FAM19A2 sequence (Q8N3H0), the signal sequence is underlined and the N-terminal sequence is boxed. After protein translation, the signal sequence is cleaved, and the sequence within the box is thought to be the N-terminal sequence of FAM19A2. [Figure 3] The graph shows the results of measuring the titer of anti-FAM19A2 antibodies using rFAM19A2 solid-phase ELISA. The vertical axis indicates absorbance, and the horizontal axis indicates the concentration of anti-FAM19A2 antibody used. In the range of 4 ng / mL to 500 ng / mL of anti-FAM19A2 antibody, five anti-FAM19A2 antibody clones showed an absorbance of 0.3 or higher. Therefore, the five anti-FAM19A2 antibody clones reacted strongly with rFAM19A2. Furthermore, no differences were observed between the clones. [Figure 4]The graph shows the results of epitope analysis of anti-FAM19A2 antibodies using three peptides. The vertical axis indicates absorbance. Three clones, A2P1D11, A2P3H12, and A2P12D4, reacted with peptide 1, peptide 2, and peptide 3. On the other hand, A2P13H8 did not react with peptide 3, and FA2G2H9 did not react with any peptide. Therefore, the antibody epitopes were classified into the A2P1D11, A2P3H12, and A2P12D4 groups, which recognize the epitope (ANHHKAHH) that is part of the N-terminal sequence of FAM19A2; the A2P13H8 group, which recognizes the ANHHKAHHVKH sequence but not ANHHKAHH; and the FA2G2H9 group, which does not recognize any peptide. [Figure 5] The results of immunostaining FAM19A2-expressing cells using anti-FAM19A2 antibodies are shown. The top photograph (FAM19A2) shows the results of staining using anti-FAM19A2 antibodies. The bottom photograph (Merge) shows the results of staining the cell nuclei (DAPI staining) overlaid with the results shown in the top photograph. Staining with A2P1D11, A2P3H12, A2P12D4, A2P13H8, and FA2G2H9 was confirmed in the human FAM19A2 stable expression cell line OE2-7. No staining was observed in the control cell line CN2. It was found that all five of the obtained clones were stained with anti-FAM19A2 antibodies by cell staining. [Figure 6] These figures show the results of immunostaining tumor and non-tumor areas of follicular carcinoma using anti-FAM19A2 antibody. The top photograph shows the results of staining the tumor area of ​​follicular carcinoma. The bottom photograph shows the results of staining the non-tumor area of ​​follicular carcinoma. Staining of the tumor area of ​​follicular carcinoma was confirmed with A2P3H12, A2P1D11, and A2P12D4. However, A2P13H8 and FA2G2H9 did not stain the tumor area of ​​follicular carcinoma. Neither clone stained the non-tumor area. These results indicate that some anti-FAM19A2 antibodies are unable to stain follicular carcinoma, and that antibodies that recognize an epitope that is part of the N-terminal sequence of FAM19A2 can stain follicular carcinoma. [Figure 7]These figures show the results of immunostaining the tumor and non-tumor regions of conventional papillary carcinoma using the anti-FAM19A2 antibody. The top photograph shows the results of staining the tumor region of conventional papillary carcinoma. The bottom photograph shows the results of staining the non-tumor region of conventional papillary carcinoma. Staining of the tumor region of conventional papillary carcinoma was confirmed with A2P3H12, A2P1D11, and A2P12D4. However, A2P13H8 and FA2G2H9 did not stain the tumor region of conventional papillary carcinoma. Neither clone stained the non-tumor region. These results demonstrate that some anti-FAM19A2 antibodies cannot stain conventional papillary carcinoma, and that antibodies that recognize an epitope that is part of the N-terminal sequence of FAM19A2 can stain conventional papillary carcinoma. [Figure 8] These figures show the results of immunostaining of tumor and non-tumor tissue of follicular papillary carcinoma using anti-FAM19A2 antibody. The top photograph shows the results of staining of tumor tissue of follicular papillary carcinoma. The bottom photograph shows the results of staining of non-tumor tissue of follicular papillary carcinoma. Staining of tumor tissue of follicular papillary carcinoma was confirmed with A2P3H12, A2P1D11, and A2P12D4. However, A2P13H8 and FA2G2H9 did not stain the tumor tissue of follicular papillary carcinoma. Neither clone stained the non-tumor tissue. These results demonstrate that some anti-FAM19A2 antibodies cannot stain follicular papillary carcinoma, and that antibodies that recognize an epitope that is part of the N-terminal sequence of FAM19A2 can stain follicular papillary carcinoma. DETAILED DESCRIPTION OF THE INVENTION

[0011] Specific embodiments for carrying out the present invention will be described below. The following description is intended to facilitate understanding of the invention and is not intended to limit the scope of the present invention.

[0012] 1.Definition As used herein, the term "antibody" refers to a protein comprising at least one immunoglobulin variable domain. For example, an antibody may comprise one heavy chain variable region (VH) and one light chain variable region (VL). For example, an antibody may comprise two heavy chain variable regions and two light chain variable regions. For example, an antibody may or may not comprise a constant region.

[0013] The term "binding fragment" as used herein includes, for example, Fab, Fab', F(ab')2, and Fv. Furthermore, "binding fragment" may also include recombinantly produced binders or functional equivalents (e.g., scFv (single-chain Fv), diabody, scDb, tandem scFv, leucine zipper, sc(Fv)2 (single-chain Fv)2, etc., or other antibody portions). For example, Fab can be obtained by digesting an antibody with papain. Alternatively, F(ab')2 can be obtained by digesting an antibody with pepsin, and Fab' can be obtained by further reduction. Such antibody binding fragments can be used herein. In scFv, VL and VH are linked by an artificial polypeptide linker, so that the same antigen specificity as the original antibody can be maintained. VL and VH can be linked in the order of VH and VL or VL and VH from the N-terminus. The linker can be approximately 10 to 25 amino acids in length. The linker may be rich in glycine or may contain amino acids such as serine and threonine to enhance water solubility.

[0014] Antibodies may have the structural characteristics of IgA, IgG, IgE, IgD, IgM (and their subtypes).

[0015] Antibodies may be derived from any source, including, but not limited to, mammalian antibodies. For example, antibodies may be derived from non-human or human sources. For example, antibodies may be derived from any source, including mouse, rat, goat, pig, or primate (human and non-human primate) antibodies. Antibodies may or may not include humanized antibodies. For example, a humanized antibody may contain a variable region of non-human origin, with the remainder of the region derived from humans. For example, in a humanized antibody, only the six CDRs may be of non-human origin, and the remaining regions may be of human origin. In another example, in a humanized antibody, only the variable region (i.e., VL and VH) may be of non-human origin, and the remaining regions (e.g., constant region) may be of human origin.

[0016] The antibody may be a monoclonal antibody or a polyclonal antibody.

[0017] Antibodies and binding fragments thereof may have modifying groups. Examples of modifying groups include, but are not limited to, any one or more of the following: fluorescent molecules (e.g., FITC, Cy3, Cy5, chlorophyll, coumarin, fluorescein, rhodamine, Alexa Fluor); Fluor (registered trademark), fluorescent proteins (e.g., green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), blue fluorescent protein (CFP), orange fluorescent protein (OFP), other GFP variants (e.g., enhanced green fluorescent protein (EGFP)), RFP variants (mCherry), luciferase), enzymes that contribute to color development (e.g., peroxidase (HRP), alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, catalase, apoglucose oxidase, urease, luciferase, acetylcholinesterase), biotin, biotin-binding proteins (e.g., avidin, streptavidin, etc.), radioisotopes (e.g., 3 H,14 C. 125 I, 131 I) Or a substance that develops color due to the action of the enzyme.

[0018] As used herein, the term "detection" refers to detecting the presence of a particular substance to thereby discover the possibility of a particular disease.

[0019] As used herein, the term "diagnosis" refers to determining whether a subject has a particular condition. To make such a determination, diagnosing may include the above-mentioned detection and, in some cases, may include performing other testing steps (e.g., thyroid ultrasound, fine needle aspiration cytology, etc.).

[0020] As used herein, the term "treatment" may include ameliorating a particular symptom. Alternatively, the term "treatment" may include delaying the progression of a particular symptom. Particularly with respect to thyroid cancer, the term "treatment" may include drug administration, surgical removal, radioactive iodine therapy, and combinations thereof. Furthermore, whether or not symptoms have improved or whether or not the progression of symptoms has been delayed may be determined by comparing a group receiving treatment with a group not receiving treatment and determining whether or not there is a statistically significant difference.

[0021] The term "therapeutically effective amount" as used herein refers to the dosage of a drug, and means an amount sufficient to treat a specific symptom when administered to a subject in need of such treatment. The amount of the drug that would correspond to such an amount will vary depending on factors such as the characteristics of the drug, the state of the disease, and the characteristics of the subject in need of treatment (e.g., body weight, presence or absence of other concurrent symptoms), but can be routinely determined by one skilled in the art.

[0022] In the Japanese medical field, the term "cancer" is written in two ways: in kanji and in hiragana, and the two have different meanings. The former refers to malignant tumors derived from epithelial cells, while the latter refers to all malignant tumors. However, in this specification, the kanji and hiragana spellings of "cancer" encompass both meanings and are used interchangeably.

[0023] Hereinafter, several embodiments will be described primarily with reference to antibodies. However, the content of the embodiments described below is equally applicable to antibodies as well as binding fragments thereof. Thus, the present disclosure encompasses the content of the embodiments described below in which antibodies are replaced with binding fragments thereof.

[0024] 2.Antibody that binds to ANHHKAHH In one embodiment, the present disclosure relates to antibodies and binding fragments thereof that bind to a peptide represented by the amino acid sequence ANHHKAHH (SEQ ID NO: 1) (e.g., recognize at least a portion of this amino acid sequence (e.g., the N-terminal ANH or a sequence of greater length) as an epitope, recognize the entire amino acid sequence as an epitope, and / or specifically bind to a peptide represented by this amino acid sequence). In a further embodiment, the antibodies and binding fragments thereof have the following characteristics: (1) Binds to a cleavage fragment of FAM19A2 having the amino acid sequence ANHHKAHH (SEQ ID NO: 1) (e.g., at least a portion of the amino acid sequence of FAM19A2 (e.g., the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 6)). (2) It does not bind to a cleavage fragment of FAM19A2 that does not have the amino acid sequence ANHHKAHH (SEQ ID NO: 1).

[0025] Antibodies and binding fragments thereof may or may not contain modifying groups as described above under "1. Definitions."

[0026] In a preferred embodiment, the antibodies and binding fragments thereof may be antibodies that bind to human FAM19A2.

[0027] Thyroid cancer includes differentiated thyroid cancer, etc. Differentiated thyroid cancer includes papillary carcinoma, follicular carcinoma, etc. Furthermore, papillary carcinoma includes conventional papillary carcinoma, follicular papillary carcinoma, etc. Papillary carcinoma accounts for approximately 90% of thyroid cancers. Follicular carcinoma accounts for approximately 5% of thyroid cancers. Follicular adenoma is also a benign tumor of the thyroid gland. It is difficult to distinguish between follicular carcinoma and follicular adenoma.

[0028] The present inventors prepared several anti-FAM19A2 antibodies. Analysis of these antibodies revealed that some antibodies were positive in follicular carcinoma, while others were negative in follicular carcinoma. Furthermore, epitope analysis of the antibodies was performed. As a result, the present inventors found that anti-FAM19A2 antibodies that recognize specific epitopes exhibited follicular carcinoma-specific positivity. Furthermore, staining with anti-FAM19A2 antibodies that recognize specific epitopes showed strong positivity in tumorous areas of follicular carcinoma and negative in non-tumorous areas.

[0029] Although the following explanation is not intended to limit the scope of the invention, one hypothesis to explain the effects of the present invention is that FAM19A2 exists in several isoforms due to differences in splicing, and that a specific isoform of FAM19A2 is thought to be correlated with follicular carcinoma.

[0030] Furthermore, as a result of further research by the present inventors, some of the above-mentioned anti-FAM19A2 antibodies were found to be positive in papillary carcinoma (specifically, conventional papillary carcinoma and follicular papillary carcinoma). Specifically, these antibodies were found to be positive in the tumorous areas of papillary carcinoma and negative in the non-tumorous areas.

[0031] These follicular and papillary carcinomas are known as representative types of differentiated thyroid cancer.

[0032] Therefore, in one embodiment, the antibodies and binding fragments thereof of the present disclosure are useful for distinguishing between differentiated thyroid cancer (e.g., follicular carcinoma, papillary carcinoma, etc.) and other portions (e.g., follicular adenoma, other thyroid cancers (e.g., cancers other than papillary carcinoma and follicular carcinoma), normal cells, etc.).

[0033] In one embodiment, the antibodies and binding fragments thereof of the present disclosure are useful for any of the following applications or a combination of these applications:

[0034] (A) To detect differentiated thyroid cancer in the thyroid gland (e.g., by immunohistochemical or immunohistofluorescent staining). (B) Diagnosing whether a subject has differentiated thyroid cancer in the thyroid gland (C) Treating differentiated thyroid cancer in a subject who has or is suspected of having differentiated thyroid cancer in the thyroid gland (D) To investigate the recovery status of subjects who have received treatment for differentiated thyroid cancer. (E) Producing a composition for use in any of (A) to (D) above (F) Producing a kit for use in any of the above (A) to (D)

[0035] 3. Compositions and Kits In one embodiment, the present disclosure relates to a composition comprising the above-described antibody or binding fragment thereof, which can be used in any of (A) to (D) above.

[0036] The composition may contain components other than the above-described composition containing an antibody or a binding fragment thereof. For example, it may contain components known in the art that are suitable for storing antibodies. For example, the composition may contain water as a solvent. For example, the composition may or may not contain glycerol for purposes such as cryoprotection. For example, the composition may or may not contain sodium azide for purposes such as preservation. For example, the composition may or may not contain thyromersal for purposes such as preservation. For example, the composition may or may not contain BSA for purposes such as protein stabilization. For example, the composition may or may not contain one or more of the components that make up Tris-Buffered Saline (TBS) for purposes such as protein stabilization.

[0037] In one embodiment, the present disclosure relates to a kit comprising the above-described antibody or binding fragment thereof. The kit can be used in any of the above-described (A) to (D). In the kit, the above-described antibody or binding fragment thereof may be provided in the form of a composition. The kit may or may not include additional reagents for detecting binding between the above-described antibody or binding fragment thereof and an antigen. For example, the kit may or may not include a secondary antibody that recognizes the above-described antibody or binding fragment thereof. The secondary antibody may or may not have a modifying group as described in "1. Definitions" above. The kit may or may not include a reagent for fixing tissues or cells (e.g., paraformaldehyde, etc.). If the secondary antibody is fused to an enzyme protein (e.g., HRP, alkaline phosphatase, etc.), the kit may also include a reagent that serves as a substrate for the enzyme. The secondary antibody may also be fused to a fluorescent molecule instead of the enzyme protein. The kit may also include instructions describing procedures for using the above-described reagents.

[0038] The above-mentioned kit may be a kit for immunostaining (e.g., for immunohistochemical staining or immunohistofluorescence staining). Alternatively, the above-mentioned kit may be a kit for ELISA. In the case of an ELISA kit, the kit may include a plate having an antibody immobilized on the bottom of the wells, and an antibody recognizing an epitope different from that of the above-mentioned antibody or binding fragment thereof may be immobilized on the bottom of the wells of the plate.

[0039] 4. Method In one embodiment, the present disclosure relates to various methods using the antibodies or binding fragments thereof described above.

[0040] In one embodiment, the methods of the present disclosure relate to any one or more of the following: Methods for detecting differentiated thyroid cancer Method for detecting differentiated thyroid cancer cells Method for diagnosing differentiated thyroid cancer Method for obtaining preliminary information for diagnosing differentiated thyroid cancer Methods for determining the pathology of suspected differentiated thyroid cancer Method for predicting differentiated thyroid cancer or method for predicting cancer How to decide on a treatment strategy for differentiated thyroid cancer (e.g., whether to proceed with observation or thyroidectomy) Methods for Treating Differentiated Thyroid Cancer Methods for monitoring the condition after treatment of differentiated thyroid cancer (e.g., monitoring the condition after thyroidectomy)

[0041] In one embodiment, the method of the present disclosure may be an in vitro method. In one embodiment, the method of the present disclosure may not include a step of diagnosing a human. In one embodiment, the method of the present disclosure may not include a step of treating a human (e.g., surgery, administration of a drug, radiation therapy, etc.).

[0042] In the following, some further specific examples of the method of the present disclosure will be described in one embodiment.

[0043] 4-1.How to detect differentiated thyroid cancer in the thyroid gland In one embodiment, the present disclosure relates to a method for detecting differentiated thyroid cancer in the thyroid gland, the method being an in vitro method, and comprising detecting the presence of FAM19A2 having the amino acid sequence ANHHKAHH (SEQ ID NO: 1) in a biological sample removed from a subject using the above-described antibody or binding fragment thereof. For example, the detecting step may include contacting the biological sample with the above-described antibody or binding fragment thereof and detecting binding of FAM19A2 having the amino acid sequence ANHHKAHH (SEQ ID NO: 1) to the antibody or binding fragment thereof.

[0044] The biological sample may also be a biological sample removed from a subject's body. The biological sample may be at least a portion of thyroid tissue. The biological sample may also be, for example, at least a portion of thyroid tissue removed by surgery. The biological sample may also be one or more cells constituting at least a portion of thyroid tissue. The biological sample may also be blood, or plasma or serum separated from the blood.

[0045] The subject from whom the biological sample is collected and / or the subject from whom differentiated thyroid cancer of the thyroid is detected may be a patient with or suspected of having differentiated thyroid cancer.

[0046] The detecting step may include immunohistochemical staining or immunohistofluorescence staining.

[0047] For example, immunohistochemical or immunohistofluorescence staining procedures may include performing the following steps. Fixing the tissue (e.g., with paraformaldehyde) Paraffin embedding and sectioning Deparaffinization Activation treatment administering the above-described antibody or its binding fragment to promote binding to the target FAM19A2 in the tissue; administering a second antibody to stimulate binding with the antibody or its binding fragment; Detect the presence of the secondary antibody using an enzyme or fluorescent label fused to the secondary antibody (e.g., color development by enzymatic degradation or detection by fluorescence microscopy)

[0048] When an enzyme or fluorescent label is fused to the primary antibody, the use of the secondary antibody may be omitted.

[0049] For example, when the biological sample is blood, or plasma or serum separated from the blood, the detecting step may include treatment by ELISA.

[0050] 4-2. How to diagnose differentiated thyroid cancer In one embodiment, the present disclosure relates to a method for diagnosing differentiated thyroid carcinoma of the thyroid gland, the method comprising carrying out any one or more of the steps described in "4-1. Method for detecting differentiated thyroid carcinoma of the thyroid gland."

[0051] The subject to be diagnosed may be a patient with or suspected of having differentiated thyroid cancer.

[0052] The method includes the following steps: Obtaining a biological sample (e.g., at least a portion of thyroid tissue, blood, plasma, serum, etc.) from a subject. detecting the presence of FAM19A2 in the biological sample by contacting the biological sample with the antibody or binding fragment thereof described above and detecting binding of FAM19A2 having the amino acid sequence ANHHKAHH (SEQ ID NO: 1) to the antibody or binding fragment thereof; If FAM19A2 is detected, the subject is diagnosed with differentiated thyroid cancer.

[0053] The step of diagnosing the subject as having differentiated thyroid cancer may include making a diagnosis based on the degree of binding between the antibody and the epitope (for example, the degree of staining, fluorescence intensity, etc.).

[0054] The method for determining whether the result is positive is not limited, but can be determined, for example, by the following procedure. -The target is staining and staining intensity in the tumor area. The score is classified according to the criteria for staining patterns (Table 1), with a score of 2+ being considered positive.

[0055] [Table 1]

[0056] The method for determining a positive result may be to distinguish between any two or more of the following: ·Normal tissue ·Thyroid differentiated carcinoma tissue Non-differentiated thyroid cancer tissue (e.g., thyroid cancer other than differentiated thyroid cancer, benign goiter, benign follicular adenoma)

[0057] 4-3. Methods for treating differentiated thyroid cancer In one embodiment, the present disclosure relates to a method for treating differentiated thyroid carcinoma of the thyroid gland, the method comprising performing any one or more of the steps described in "4-1. Method for detecting differentiated thyroid carcinoma of the thyroid gland." The method comprises performing any one or more of the steps described in "4-2. Method for diagnosing differentiated thyroid carcinoma of the thyroid gland."

[0058] The subject to be treated may be a patient with or suspected of having differentiated thyroid cancer.

[0059] The method includes the following steps: Obtaining a biological sample (e.g., at least a portion of thyroid tissue, blood, plasma, serum, etc.) from a subject. detecting the presence of FAM19A2 in the biological sample by contacting the biological sample with the antibody or binding fragment thereof described above and detecting binding of FAM19A2 having the amino acid sequence ANHHKAHH (SEQ ID NO: 1) to the antibody or binding fragment thereof; If FAM19A2 is detected, the subject is diagnosed with differentiated thyroid cancer. administering to the subject a therapeutically effective amount of a therapeutic agent for differentiated thyroid cancer.

[0060] The therapeutic agent may include, for example, any one or more of the following: a radioactive iodine therapeutic agent, a molecularly targeted agent (e.g., lenvatinib, sorafenib), and the like.

[0061] In one embodiment, the step of administering a therapeutic agent may include administering a therapeutically effective amount of an antibody or binding fragment thereof capable of binding to the peptide represented by the amino acid sequence ANHHKAHH (SEQ ID NO: 1). For example, the above-described antibody or binding fragment thereof may function as a drug delivery device. For example, it may be fused to any of the above-described therapeutic agents and administered.

[0062] 4-4.Other In some diagnostic examples, follicular carcinoma may be diagnosed by confirming at least one of membranous invasion, vascular invasion, and extrathyroidal metastasis. Among these, membranous invasion and vascular invasion are known to be difficult to distinguish, and it is known to take a long time (up to 10 years) to obtain a definitive diagnosis. However, the above-described antibody and / or method, etc., in one embodiment, makes it possible to confirm the localization of FAM19A2 in tissue. In particular, the above-described antibody and / or method, etc., in one embodiment, can confirm particularly strong staining in invasive areas. Therefore, the above-described antibody and / or method, etc., in one embodiment, can aid in the diagnosis of follicular carcinoma.

[0063] 5. Supplementary Notes As described above, in one embodiment, the present disclosure relates to an anti-FAM19A2 antibody and its applications. Another known method for detecting the presence of FAM19A2 is to detect the mRNA of FAM19A2. However, various regulations may occur during the process of transcription from DNA to mRNA and translation from mRNA to protein. For example, even if the same amount of mRNA is present, various factors may enhance or inhibit the protein translation process. Therefore, in extreme cases, even if the amount of mRNA is low, a large amount of the corresponding protein may be produced, while conversely, even if the amount of mRNA is high, only a small amount of the corresponding protein may be produced. From this perspective, methods based on anti-FAM19A2 antibodies and their applications are advantageous in that they allow for more direct detection of target substances compared to methods that detect mRNA. [Example]

[0064] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0065] Example 1: Preparation of mouse anti-human recombinant FAM19A2 monoclonal antibody As shown in Figure 1, the human FAM19A2 sequence (Q8N3H0) was inserted into an expression vector containing an ampicillin-resistant T7 promoter and transformed into Escherichia coli strain BL21(DE3), where expression was induced with IPTG. The soluble fraction was collected by sonication, and the histidine-tagged recombinant protein (rFAM19A2-EGFP) was purified using a Ni column. BALB / c mice were immunized with rFAM19A2-EGFP multiple times every 2 weeks. A 1 mg / mL emulsion was prepared using Freund's complete adjuvant (FCA) for the first immunization and Freund's incomplete adjuvant (FIA) for subsequent immunizations. The rFAM19A2-EGFP emulsion was administered intraperitoneally at 100 μg / head for the first immunization and 50 μg / head for subsequent immunizations. One week after immunization, blood was collected from the tail vein and serum antibody titers were measured using human recombinant FAM19A2 (rFAM19A2, R&D 4179-TA) solid-phase ELISA (1, detailed procedures are described below). Individuals with confirmed elevated rFAM19A2 antibody titers were intraperitoneally administered rFAM19A2-EGFP diluted with saline (boost). Three days after the boost, mouse splenocytes were collected and fused with myeloma cells by electrofusion. Approximately one week after cell fusion, rFAM19A2-positive hybridomas were selected using rFAM19A2 solid-phase ELISA and single-cloned to establish FA2G2H9. BALB / c mice were intraperitoneally administered 500 μL of pristane (1921-70-6, Fujifilm Wako Pure Chemical Industries), and after recovery, FA2G2H9 hybridomas were transfected at 3 × 10 6 The mouse ascites was intraperitoneally administered at 100 cells / head, and ascites fluid was collected. IgG monoclonal antibodies were purified from the mouse ascites using protein A affinity column chromatography. The rFAM19A2 antibody titer of the IgG antibody was confirmed by rFAM19A2 solid-phase ELISA. Purified antibodies against FA2G2H9 were obtained.

[0066] Example 2: Preparation of mouse anti-human FAM19A2 peptide monoclonal antibody A peptide containing the N-terminal sequence of human FAM19A2 (synthesis outsourced to PH Japan Co., Ltd.; peptide containing the N-terminal sequence: ANHHKAHHVKTGTC) was conjugated with Imject Blue Carrier Protein (Product Code: 77660, Thermo Scientific) (BCIP-FAM19A2pep) and immunized with this conjugate every 2 weeks into BALB / c mice. Figure 2 shows the human FAM19A2 sequence (Q8N3H0) and its N-terminal sequence (boxed). A 1 mg / mL emulsion was prepared using Freund's complete adjuvant (FCA) for the first immunization and Freund's incomplete adjuvant (FIA) for subsequent immunizations. The BCIP-FAM19A2pep emulsion was administered intraperitoneally at 100 μg / head for the first immunization and 50 μg / head for subsequent immunizations. One week after immunization, blood was collected from the tail vein and serum antibody titers were measured using human recombinant FAM19A2 (rFAM19A2, R&D 4179-TA) solid-phase ELISA (1, detailed procedures described below). Individuals with confirmed elevated rFAM19A2 antibody titers were given a boost intraperitoneally with saline-diluted BCIP-FAM19A2 pep. Three days after the boost, mouse splenocytes were collected and fused with myeloma cells by electrofusion. Approximately one week after cell fusion, hybridomas positive for rFAM19A2 or the peptide containing the human FAM19A2 N-terminal sequence were selected using rFAM19A2 solid-phase ELISA or cell sorting using a biotin-labeled FAM19A2 N-terminal sequence peptide (2, detailed procedures described below). These hybridomas were then single cloned and established into cell lines. A total of four clones (A2P1D11, A2P3H12, A2P12D4, and A2P13H8) were established in a similar manner. 500 μL of pristane (1921-70-6, Fujifilm Wako Pure Chemical Industries) was intraperitoneally administered to BALB / c mice, and after recovery, the four hybridoma clones (A2P1D11, A2P3H12, A2P12D4, and A2P13H8) were cultured at 3 × 10 6The mouse ascites was intraperitoneally administered at 100 cells / head, and ascites fluid was collected. IgG monoclonal antibodies were purified from the mouse ascites using protein A affinity column chromatography. The rFAM19A2 antibody titer of the IgG antibody was confirmed using rFAM19A2 solid-phase ELISA. Purified antibodies of four mouse anti-human FAM19A2 monoclonal antibody clones (A2P1D11, A2P3H12, A2P12D4, and A2P13H8) were obtained.

[0067] The above-mentioned rFAM19A2 solid-phase ELISA assay was carried out according to the following procedure.

[0068] rFAM19A2 solid phase ELISA measurement method Recombinant human TAFA2 / FAM19A2 protein (NOVUS, NBP2-35034) was diluted to 0.5 μg / mL in PBS and immobilized overnight at 50 μl / well. 300 μl / well of 1% BSA / 5% sucrose / PBS (blocking solution) was added and incubated at room temperature for 1 hour. After washing with 0.05% Tween / PBS (PBST), 50 μl / well of 4–500 ng / mL IgG antibody was added. After incubation at room temperature for 1 hour, the plate was washed with PBST and horseradish peroxidase-labeled anti-mouse IgG (115-035-164, Jackson) was added at 50 μl / well. After reacting at room temperature for 1 hour, the plate was washed with PBST, and 50 μl / well of 3,3',5,5'-tetramethylbenzidine color-developing solution was added. The color-developing reaction was stopped by adding 50 μg / well of 1N sulfuric acid, which had been reacted at room temperature for 20 minutes, and the absorbance at 450 nm was measured.

[0069] The results are shown in Figure 3. Within the rFAM19A2 concentration range of 4 ng / mL to 500 ng / mL, five anti-FAM19A2 antibody clones (one clone obtained in Example 1 and four clones obtained in Example 2) showed absorbances of 0.3 or higher. Thus, the five anti-FAM19A2 antibody clones reacted strongly with rFAM19A2. Furthermore, no differences in antibody titer were observed among the clones.

[0070] The above-mentioned cell sorting using a peptide having the N-terminal sequence of biotin-labeled FAM19A2 was carried out according to the following procedure.

[0071] Cell sorting method using a peptide containing the N-terminal sequence of biotin-labeled FAM19A2 1 μg of biotin-labeled peptide with the N-terminal sequence of FAM19A2 (ANHHKAHHVKTGTK-Biotin, synthesis outsourced to PH Japan Co., Ltd.) was mixed with 100 μL of streptavidin PE-labeled (2626020, SONY). In a separate tube, 1 μg of biotin-labeled peptide with the N-terminal sequence of FAM19A2 was mixed with 40 μL of streptavidin FITC-labeled (2626010, SONY). 200 μg of albumin and biotin-labeled bovine (A8549, Sigma) were added to each. 2 x 10 6 Hybridoma cells were added. After 30 minutes at room temperature, the cells were centrifuged at 1,000 rpm for 5 minutes and suspended in 1 mL of HBSS / 25 mM HEPES / 1 mM EDTA / 1% FBS pH 7.4 (HBSS buffer). The cells were then centrifuged twice more and washed with HBSS buffer. The cell suspension was passed through a 40 μm filter, and 3 μL of 7-AAD Viability Staining Solution (420404, Biolegend) was added. The FAM19A2-PE(+) and FAM19A2-FITC(+) fractions were sorted using a CELL SORTER SH800S (SONY).

[0072] Example 3 Epitope analysis of anti-FAM19A2 antibody Epitope analysis of five anti-FAM19A2 antibody clones (A2P1D11, A2P3H12, A2P12D4, A2P13H8, and FA2G2H9) was performed using biotin-labeled peptides containing at least a portion of the N-terminal sequence of FAM19A2 (synthesized by Biologica Co., Ltd.) (specifically, using the three types of peptides shown in Table 2).

[0073] [Table 2]

[0074] 1 μg / mL streptavidin (102-17864, Fujifilm Wako Pure Chemical Industries) was immobilized overnight at 50 μg / well on a plate (3801-096, IWAKI). After washing with PBST, 50 μg / well of the three types of biotin-labeled peptides shown in Table 2 were added. After incubation at room temperature for 1 hour, the plate was washed with PBST. Five mouse anti-FAM19A2 antibody clones (5 clones) were added at 0.1–10 μg / mL at 50 μg / well. After incubation at room temperature for 1 hour, the plate was washed with PBST and goat horseradish peroxidase-labeled anti-mouse IgG (115-035-164, Jackson) was added. After incubation at room temperature for 1 hour, the plate was washed with PBST and 3,3',5,5'-tetramethylbenzidine color developer solution was added at 50 μg / well. After reacting at room temperature for 20 minutes, 1N sulfuric acid was added at 50 μg / well to stop the color reaction, and the absorbance at 450 nm was measured.

[0075] The results are shown in Figure 4. Three clones, A2P1D11, A2P3H12, and A2P12D4, reacted with peptide 1, peptide 2, and peptide 3. On the other hand, A2P13H8 did not react with peptide 3, and FA2G2H9 did not react with any of the peptides.

[0076] As a result of the above, the antibody epitopes were classified as follows: A2P1D11, A2P3H12, and A2P12D4 groups that recognize the N-terminal portion (ANHHKAHH, SEQ ID NO: 1) of the N-terminal sequence of FAM19A2 A2P13H8 group that recognizes the central part (HKAHHVKT, SEQ ID NO: 5) of the N-terminal sequence of FAM19A2; FA2G2H9 group that does not recognize any peptides

[0077] Example 4 Cell staining using a follicular carcinoma cell line-derived human FAM19A2 stably expressing cell line The follicular carcinoma cell line FTC-238 (provided by the European Collection of Authenticated Cell Cultures (ECACC)) was transfected with an expression vector (pcDNA3-hFAM19A2-Gs linker-Flag tag-His tag), and the stable human FAM19A2 cell line, OE2-7, was established through drug resistance screening with G418 (108321-42-2, Fujifilm Wako Pure Chemical Industries). FAM19A2 mRNA expression was confirmed by RT-PCR. Cells were stained with 5 μg / mL mouse anti-FAM19A2 antibodies (FA2G2H9, A2P1D11, A2P3H12, A2P12D4, A2P13H8) as the primary antibody and 5 μg / mL red fluorescently labeled anti-mouse IgG antibody (Alexa Fluor 594) as the secondary antibody, and observed under a fluorescence microscope. The cells were directly fixed with paraformaldehyde (PFA) and then permeabilized with Triton X-100.

[0078] The results of cell staining are shown in Figure 5. Staining with A2P1D11, A2P3H12, A2P12D4, A2P13H8, and FA2G2H9 was confirmed in the human FAM19A2 stable expression cell line OE2-7. All five of the obtained anti-FAM19A2 antibodies were found to be stainable by cell staining. The immunostaining results are summarized in Table 3. The results are shown in two levels depending on whether or not staining occurred. Stained: +, Not stained: - [Table 3]

[0079] Example 5 FAM19A2 tissue staining using FFPE specimens of follicular carcinoma Formalin-fixed paraffin-embedded tissues from two cases of follicular carcinoma with distant metastasis were stained with five clones of mouse anti-FAM19A2 antibodies (A2P1D11, A2P3H12, A2P12D4, A2P13H8, and FA2G2H9).

[0080] After deparaffinization with xylene and ethanol, the tissues were subjected to antigen retrieval with citrate buffer, pH 6.0 (autoclaved at 125°C for 5 minutes). Following the instructions for Simple stain MAX·PO(M) Nichirei (Nichirei Biosciences, 424131), the tissues were stained and examined for positive reactions under an optical microscope. Five mouse anti-FAM19A2 antibody clones were added at 50 μg / mL and incubated at room temperature for 1 hour.

[0081] Figure 6 shows the results of histological staining of follicular carcinoma. Staining of the tumorous areas of follicular carcinoma was confirmed with A2P3H12, A2P1D11, and A2P12D4. However, A2P13H8 and FA2G2H9 did not stain in the tumorous areas of follicular carcinoma. Neither clone stained in the non-tumorous areas. A2P3H12, A2P1D11, and A2P12D4 stained in two cases of follicular carcinoma, with stronger staining observed in the capsular invasive areas than in the tumorous areas. These results strongly suggest that FAM19A2 protein is expressed in follicular carcinoma.

[0082] The immunostaining results are summarized in Table 4. The staining intensity is graded into three levels. Strongly stained: 2+, Weakly stained: 1+, No staining: 0

[0083] [Table 4]

[0084] Table 4 shows that A2P13H8, which recognizes HKAHHVKT but not ANHHKAHH, and FA2G2H9, which does not react to the peptide, do not stain in the tumor area of ​​follicular carcinoma, while A2P1D11, A2P3H12, and A2P12D4, which recognize ANHHKAHH, stain in the tumor area of ​​follicular carcinoma.

[0085] Even A2P13H8 and FA2G2H9, which were confirmed to react strongly with rFAM19A2 in Example 2, did not stain follicular carcinoma, whereas A2P1D11, A2P3H12, and A2P12D4, which recognize ANHHKAHH, stained follicular carcinoma. The Examples described herein demonstrated that there are anti-FAM19A2 antibodies that cannot stain follicular carcinoma, and that antibodies that recognize ANHHKAHH, a part of the N-terminal sequence of FAM19A2, as an epitope can stain follicular carcinoma.

[0086] Example 6 FAM19A2 tissue staining using FFPE specimens of papillary carcinoma Formalin-fixed paraffin-embedded tissues from conventional and follicular papillary thyroid carcinomas were stained with five mouse anti-FAM19A2 antibody clones (A2P1D11, A2P3H12, A2P12D4, A2P13H8, and FA2G2H9).

[0087] After deparaffinization with xylene and ethanol, the tissues were subjected to antigen retrieval with citrate buffer, pH 6.0 (autoclaved at 125°C for 5 minutes). Following the instructions for Simple stain MAX·PO(M) Nichirei (Nichirei Biosciences, 424131), the tissues were stained and examined for positive reactions under an optical microscope. Five mouse anti-FAM19A2 antibody clones were added at 50 μg / mL and incubated overnight at 4°C.

[0088] Figure 7 shows the results of histological staining for conventional papillary carcinoma, and Figure 8 shows the results for follicular papillary carcinoma. In both cases, A2P3H12, A2P1D11, and A2P12D4 stained the tumor area, while A2P13H8 and FA2G2H9 did not. Neither clone stained the non-tumor area. These results strongly suggest that FAM19A2 protein is expressed in conventional papillary carcinoma and follicular papillary carcinoma.

[0089] The immunostaining results are summarized in Table 5. The staining intensity is graded into three levels. Strongly stained: 2+, Weakly stained: 1+, No staining: 0

[0090] [Table 5]

[0091] Table 5 shows that A2P13H8, which recognizes HKAHHVKT but not ANHHKAHH, and FA2G2H9, which does not react to the peptide, do not stain in the tumor area of ​​papillary carcinoma, while A2P1D11, A2P3H12, and A2P12D4, which recognize ANHHKAHH, stain in the tumor area of ​​papillary carcinoma.

[0092] Specific embodiments of the invention have been described above. The above embodiments are merely illustrative examples, and the present invention is not limited to these embodiments. For example, technical features disclosed in one of the above embodiments may be applied to other embodiments. Furthermore, unless otherwise specified, for a particular method, the order of some steps may be interchanged, and additional steps may be added between two specific steps. The scope of the present invention is defined by the claims.

Claims

1. An antibody or a binding fragment thereof, which is capable of binding to a peptide represented by the amino acid sequence ANHHKAHH (SEQ ID NO: 1).

2. 2. The antibody or binding fragment thereof of claim 1, binds to a truncated fragment of FAM19A2 having the amino acid sequence ANHHKAHH (SEQ ID NO: 1); does not bind to a cleavage fragment of FAM19A2 that does not have the amino acid sequence ANHHKAHH (SEQ ID NO: 1); An antibody or binding fragment thereof.

3. The antibody or binding fragment thereof of claim 1 which is a monoclonal antibody.

4. A composition comprising the antibody or binding fragment thereof of any one of claims 1 to 3, Used to detect differentiated thyroid cancer in the thyroid gland, composition.

5. The composition of claim 4, wherein the differentiated thyroid carcinoma is follicular carcinoma.

6. The composition of claim 4, wherein the differentiated thyroid carcinoma is papillary carcinoma.

7. The composition of claim 4, which is used to detect differentiated thyroid carcinoma in the thyroid gland by immunohistochemical staining techniques.

8. A kit for detection comprising the antibody or binding fragment thereof according to any one of claims 1 to 3, Used to detect differentiated thyroid cancer in the thyroid gland, Detection kit.

9. 1. A method for detecting differentiated thyroid cancer in the thyroid gland of a subject, said method being an in vitro method comprising: The method comprises: Detecting the presence of FAM19A2 having the amino acid sequence ANHHKAHH (SEQ ID NO: 1) in a biological sample taken ex vivo from a subject using the antibody or binding fragment thereof of any one of claims 1 to 3; A method comprising:

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