KR102644178B1 - Novel TMEM165-CLOCK epitope and antibodies thereto - Google Patents

Abstract

The present invention relates to a TMEM165-CLOCK antibody epitope, an antibody or antigen-binding fragment thereof that specifically binds to the epitope, and a composition for diagnosing, preventing, or treating cancer comprising the antibody or antigen-binding fragment. The TMEM165-CLOCK antibody epitope according to the present invention is an antigen binding site of a novel sequence identified from the cancer cell-specific expression fusion gene TMEM165-CLOCK, and since the antibody against this was confirmed to have high binding affinity to TMEM165-CLOCK, the expression of TMEM165-CLOCK and It can be useful in the diagnosis, prevention, or treatment of cancer, a related disease.

Description

Novel TMEM165-CLOCK epitope and antibodies thereto {Novel TMEM165-CLOCK epitope and antibodies thereto}

The present invention relates to a TMEM165-CLOCK antibody epitope, an antibody or antigen-binding fragment thereof that specifically binds to the epitope, and a composition for diagnosing, preventing, or treating cancer comprising the antibody or antigen-binding fragment.

To date, cancer diagnosis methods include invasive methods such as tissue sample collection and endoscopy. In particular, biopsy is performed by removing part of the area suspected of having a disease and observing it under a microscope. Therefore, in order to use a needle, punch, endoscope, or laparoscope to collect tissue samples, the human body must be incised, which not only causes considerable discomfort to the patient, but also leaves scars and takes a long time to recover.

In addition, most treatments for cancer that are currently in progress are surgical resection of the lesion, and especially when the goal is complete cure, surgical resection is the only method. In such surgical resection, the principle is to resect as wide a range as possible in surgeries aimed at complete cure, but the range of resection is sometimes determined by considering the aftereffects of extensive resection after surgery. However, even in this case, if the cancer has spread to other organs, curative surgery is not possible, so in this case, other methods such as anticancer drug administration are selected. Anticancer drugs currently on the market provide temporary relief of symptoms, suppression of recurrence after resection, and survival. It only has a temporary effect of extending the period, but has limitations in treating the underlying cancer, and also causes double suffering to patients due to the side effects and economic burden of anticancer drug administration.

Therefore, in order to treat cancer, it is most important to develop a cancer diagnosis method with high sensitivity and specificity in the pre-treatment stage, and such diagnosis must be able to detect cancer in its early stages. Furthermore, customized diagnosis and treatment are required by predicting the prognosis for specific cancers.

Meanwhile, cancer gene mutations occur due to chromosomal translocation, which causes the generation of fusion genes that are expressed by fusing different genes. In other words, a fusion gene is a hybrid gene formed by chromosomal rearrangement (translocation, deletion, inversion), trans-splicing, and read-through event. Fusion genes are known to be involved in the development and activation mechanisms of various tumors. In addition, even within the same cancer type, each patient has tumor heterogeneity, and fusion genes can be helpful in developing molecular target drugs by providing a standard for distinguishing cancer diversity for personalized treatment. Therefore, the specific expression of the fusion gene can be used as useful information for predicting and diagnosing cancer or as an effective prognostic marker in the treatment of cancer.

The present inventors confirmed the specific expression of the fusion gene TMEM165-CLOCK in various sarcoma cancer tissues such as dermatofibrosarcoma, liposarcoma, undifferentiated pleomorphic sarcoma, and osteochondroma. In addition to sarcoma cancer, we confirmed the specific expression of TMEM165-CLOCK in breast cancer, prostate cancer, and lung cancer cell lines. As high expression was confirmed, it was confirmed that the fusion gene TMEM165-CLOCK could be presented as a molecular marker for cancer cells. Accordingly, the novel sequence of TMEM165-CLOCK was used to produce an epitope and an antibody that specifically binds to it. In one example, the TMEM165-CLOCK specific antibody can be used for the diagnosis, prevention, or treatment of cancer. Confirmed.

Accordingly, the object of the present invention is to provide a TMEM165-CLOCK antibody epitope containing the amino acid sequence shown in SEQ ID NO: 1 and an antibody or antigen-binding fragment thereof that specifically binds to the epitope.

Another task is to provide a composition for diagnosing cancer and a composition for preventing or treating cancer, including a TMEM165-CLOCK specific antibody or antigen-binding fragment thereof.

The present invention provides a TMEM165-CLOCK antibody epitope comprising the amino acid sequence represented by SEQ ID NO: 1.

The epitope may include a polypeptide consisting of the 300-321st amino acid sequence of the TMEM165 protein shown in SEQ ID NO: 3.

Additionally, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to the TMEM165-CLOCK antibody epitope containing the amino acid sequence shown in SEQ ID NO: 1.

The antibody or antigen-binding fragment is selected from the group consisting of Fab, Fd, Fab', dAb, F(ab'), F(ab')2, scFv, Fv, scFv dimer, diabody, and linear antibody. It may be possible.

The antibody may include the amino acid sequence represented by SEQ ID NO: 2.

The antibody may be a single chain antibody (scFv).

Additionally, the present invention provides a TMEM165-CLOCK-specific single chain antibody (scFv) or an antigen-binding fragment thereof comprising the amino acid sequence shown in SEQ ID NO: 2.

The present invention also provides a polynucleotide encoding the antibody or antigen-binding fragment thereof.

Additionally, the present invention provides a recombinant expression vector containing the above polynucleotide.

The present invention also provides a transformant transformed with the vector.

The present invention also provides a composition for diagnosing cancer, comprising the antibody or antigen-binding fragment thereof.

The cancer may be one or more types selected from the group consisting of stomach cancer, colon cancer, kidney cancer, blood cancer, sarcoma cancer, ovarian cancer, breast cancer, cervical cancer, thyroid cancer, prostate cancer, pancreatic cancer, liver cancer, and lung cancer.

The present invention also provides a composition for preventing or treating cancer, comprising the antibody or antigen-binding fragment thereof.

The cancer may be one or more types selected from the group consisting of stomach cancer, colon cancer, kidney cancer, blood cancer, sarcoma cancer, ovarian cancer, breast cancer, cervical cancer, thyroid cancer, prostate cancer, pancreatic cancer, liver cancer, and lung cancer.

The composition may further include PBMC cells or NK cells.

The present invention also provides a method of providing information on cancer diagnosis, comprising reacting the antibody or antigen-binding fragment thereof with a target sample.

The cancer may be one or more types selected from the group consisting of stomach cancer, colon cancer, kidney cancer, blood cancer, sarcoma cancer, ovarian cancer, breast cancer, cervical cancer, thyroid cancer, prostate cancer, pancreatic cancer, liver cancer, and lung cancer.

The TMEM165-CLOCK antibody epitope according to the present invention is an antigen binding site of a novel sequence identified from the cancer cell-specific expression fusion gene TMEM165-CLOCK, and since the antibody against this was confirmed to have high binding affinity to TMEM165-CLOCK, the expression of TMEM165-CLOCK and It can be useful in the diagnosis, prevention, or treatment of cancer, a related disease.

Figure 1 is a diagram showing the expression level of TMEM165-CLOCK in normal cells and various sarcoma tissues. Top image - Lanes 1 and 2: Normal tissue, Lane 3: Dermatofibrosarcoma protuberans (DFSP), Lane 4: Undifferentiated pleomorphic sarcoma (UPS), Bottom image - Lane 1: Dermatofibrosarcoma (DFSP; Dermatofibrosarcoma protuberans), Lane 2: Low-grade liposarcoma, Lane 3: Undifferentiated pleomorphic sarcoma (UPS), Lane 4: Giant cell tumor, Lane 5: Osteochondroma .
Figure 2 is a diagram showing the expression level of TMEM165-CLOCK in cancer cells other than sarcoma. Lane 1: NCI-H1299, Lane 2: NCI-H1437, Lane 3: NCI-H1793, Lane 4: HCC827, Lane 5: NCI-H23.
Figure 3 is a diagram showing the base sequence difference (a), amino acid sequence difference (b), TMEM165-CLOCK protein schematic diagram (c), and 3D structure of the synthetic peptide (d) between TMEM165 and TMEM165-CLOCK fusion.
Figure 4 is a diagram showing immunostaining of a sarcoma cell line overexpressing TMEM165-CLOCK using a TMEM165-CLOCK specific antibody.
Figure 5 shows apoptosis (a) of a sarcoma cancer cell line overexpressing TMEM165-CLOCK using immune-activated PBMC cells and a TMEM165-CLOCK-specific antibody, and cells of a sarcoma cancer cell line overexpressing TMEM165-CLOCK using immune-activated NK cells and a TMEM165-CLOCK-specific antibody. This is a diagram showing death (b).
Figure 6 shows cell proliferation of a: sarcoma cell line (KHOS/NP), b: TMEM165-CLOCK overexpression stabilization cell line (KHOS/NP_TMEM165-CLOCK), and c: lung cancer cell line (NCI-H1793) by treatment with TMEM165-CLOCK specific antibody. This diagram shows the inhibition of cell proliferation.
Figure 7 is a diagram showing tumor growth inhibition in an experimental group (TMEM165-CLOCK Ab.) treated with a TMEM165-CLOCK specific antibody in a tumor-induced mouse model. a: Mouse tumor comparison image, b: Quantification graph.

Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the technical field to which the present invention pertains.

Terms defined in commonly used dictionaries should be interpreted as having meanings consistent with the meanings they have in the context of the related technology, and should not be interpreted in an idealized or overly formal sense unless explicitly defined in the present application. No. Additionally, in describing the present invention, if it is determined that a detailed description of related known technologies may unnecessarily obscure the gist of the present invention, the detailed description will be omitted.

The present inventors discovered TMEM165-CLOCK, a significant fusion gene commonly expressed in various sarcoma tissues, based on approximately 30,000 human genes in a total of 54 tissues, including sarcoma and normal tissues.

The fusion gene was a combination of the TMEM165 gene in the forward direction (5'→3') and the CLOCK gene in the reverse direction. It was inferred that this gene could be used as a sarcoma-specific antigen and the sequence was analyzed.

TMEM165-CLOCK (Fusion in Figure 3b) is identical to the amino acid sequence of Homo sapiens transmembrane protein 165 (TMEM165 in Figure 3a) with accession number NM_018475.5 reported to NCBI from positions 1 to 299, with differences in amino acid sequences from positions 300 to 321. It was confirmed that there is. In this specification, the TMEM165-CLOCK (Fusion) sequence of FIG. 3b is indicated as SEQ ID NO: 3. The sequence GSFSPWLILHACSDGKGHLERN (SEQ ID NO: 1) of the terminal 300-321 aa of TMEM165-CLOCK was determined as an antigen-specific region, and a peptide was produced using it.

The present invention provides a TMEM165-CLOCK antibody epitope containing the amino acid sequence represented by SEQ ID NO: 1.

The TMEM165-CLOCK is of human origin, but is not limited thereto. Specifically, the epitope may include a polypeptide consisting of the 300-321st amino acid sequence of the TMEM165-CLOCK protein shown in SEQ ID NO: 3.

In the present invention, an “epitope” may be referred to as an epitope or an antigenic determinant, and is a specific part of an antigen that allows the immune system, such as antibodies, B cells, and T cells, to identify the antigen. Specifically, an epitope refers to a set of amino acid residues in the antigen binding site recognized by a specific antibody, or in T cells, residues required for recognition by the T cell receptor protein and/or Major Histocompatibility Complex (MHC) receptor. . Epitopes according to the present invention are molecules that form a site recognized by antibodies, T cell receptors, or HLA molecules, and refer to primary, secondary, and tertiary peptide structures, or charges. Additionally, isolated or purified protein or peptide molecules larger than the epitope of the present invention and containing the epitope of the present invention may be included within the scope of the present invention. In the present invention, the terms epitope or peptide may often be used interchangeably.

The present invention can provide a polynucleotide encoding the epitope. Specifically, the polynucleotide encoding the TMEM165-CLOCK epitope protein is interpreted as including a sequence showing substantial identity, considering mutations with biologically equivalent activity. The term 'substantial identity' means that when the sequence of the present invention and any other sequence are aligned to the maximum extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art, the minimum It refers to a sequence that exhibits 60% homology, more specifically 70% homology, even more specifically 80% homology, and most specifically 90% homology.

Therefore, an amino acid sequence having high homology to the sequence represented by SEQ ID NO: 1, for example, having a homology of 70% or more, specifically 80% or more, more specifically 90% or more, and the present invention Amino acid sequences that can equally function as TMEM165-CLOCK antibody epitopes should also be construed as falling within the scope of the present invention.

Additionally, a recombinant expression vector containing the polynucleotide and a transformant transformed with the vector can be provided.

In another aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to the TMEM165-CLOCK antibody epitope containing the amino acid sequence shown in SEQ ID NO: 1.

The TMEM165-CLOCK antibody epitope is as described above.

The 'antibody that specifically binds to the TMEM165-CLOCK antibody epitope' refers to an antibody that can specifically bind to and detect the TMEM165-CLOCK protein. The above antibodies may be used interchangeably with TMEM165-CLOCK specific antibodies, anti-TMEM165-CLOCK monoclonal antibodies, or TMEM165-CLOCK antibodies.

The term 'specifically binding' is the same as the meaning of "specifically recognizing" commonly known in the art, wherein the antigen and antibody specifically interact to form an antigen-antibody complex. It can form and also mean an immunological response.

The antibody or antigen-binding fragment thereof according to the present invention can preferably bind specifically to the 300-321st amino acid sequence of the TMEM165-CLOCK protein represented by SEQ ID NO: 3, that is, the amino acid sequence represented by SEQ ID NO: 1. Therefore, the antibody or antigen-binding fragment thereof according to the present invention can be used for the purpose of detecting or specifically targeting TMEM165-CLOCK.

In the present invention, antibodies include not only whole antibodies but also functional fragments of antibody molecules that retain antigen-binding function. Examples of the functional fragment specifically include (i) a Fab fragment consisting of the variable region of the light chain (VL) and the variable region of the heavy chain (VH), the constant region of the light chain (CL), and the first constant region of the heavy chain (CH1); (ii) Fd fragment consisting of VH and CH1 domains; (iii) an Fv fragment consisting of the VL and VH domains of a single antibody; (iv) dAb fragment consisting of VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragment, a bivalent fragment containing two linked Fab fragments; (vii) a single chain Fv molecule (scFv) linked by a peptide linker that joins the VH domain and the VL domain to form an antigen binding site; (viii) bispecific single-chain Fv dimers and (ix) diabodies, which are multivalent or multispecific fragments produced by gene fusion.

A full antibody has a structure of two full-length light chains and two full-length heavy chains, and each light chain is connected to the heavy chain by a disulfide bond. The antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fvs (scFV), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFV) ) and anti-idiotype (anti-Id) antibodies, and epitope-binding fragments of the above antibodies.

The antibody or antigen-binding fragment is selected from the group consisting of Fab, Fd, Fab', dAb, F(ab'), F(ab')2, scFv, Fv, scFv dimer, diabody, and linear antibody. It may be one, but is not limited thereto.

In the present invention, the term 'monoclonal antibody' is a term known in the art and refers to a highly specific antibody directed against a single antigenic site. Monoclonal antibodies are also called monoclonal antibodies, monoclonal antibodies, or monoclonal antibodies. Unlike polyclonal antibodies, which typically contain different antibodies directed against different epitopes (antigenic determinants), monoclonal antibodies are directed against a single determinant on an antigen. Monoclonal antibodies have the advantage of improving the selectivity and specificity of diagnostic and analytical methods using antigen-antibody binding, and also have the additional advantage of not being contaminated by other immunoglobulins because they are synthesized by hybridoma culture.

In the present invention, the anti-TMEM165-CLOCK monoclonal antibody can be prepared through various methods known in the art. As an example, it may be manufactured by a chemical peptide synthesis method, or the gene encoding the monoclonal antibody may be amplified by PCR (polymerase chain reaction) or synthesized by a known method and then cloned into an expression vector and expressed. , it can also be produced using a hybridoma cell line derived from lymphocytes obtained by injecting the immunogen of the monoclonal antibody into an animal.

According to one embodiment of the present invention, a TMEM165-CLOCK specific antibody was established using phage display technology. TMEM165-CLOCK, a fusion gene confirmed to be commonly expressed in various sarcoma tissues, was cloned to purify the TMEM165-CLOCK protein, reacted with a human scFv antibody phage surface display library, and scFv-phage that specifically bound to TMEM165-CLOCK was extracted and E- Biopanning was performed to infect and amplify coli. The term 'Bio-panning' refers to the property of binding to target molecules such as antigens (viral envelope proteins), enzymes, and cell surface receptors from a phage library that displays peptides on the outer wall (coat) of the phage. This refers to the process of selecting only phages that express peptides on their surface. After performing this panning process, monoclonal antibodies were selected from the phage antibody group with high binding ability, and ELISA analysis was performed to select antibodies with excellent binding ability once more. After obtaining the phagemid DNA of the selected monoclonal antibody, sequence analysis was performed using 5'- and 3'-oligonucleotides of single chain Fv (scFv), and the common regions of the sequences read in both directions were synthesized to produce the sequence shown in SEQ ID NO: 2. TMEM165_CLOCK antibody sequence was established.

The antibody according to the present invention is not limited thereto, but may include the amino acid sequence represented by SEQ ID NO: 2, and may preferably be a single chain antibody (scFv). Also, preferably, the antibody of the present invention may be a TMEM165-CLOCK-specific single chain antibody (scFv) containing the amino acid sequence shown in SEQ ID NO: 2 or an antigen-binding fragment thereof.

The 'scFv (single chain fragment variable)' refers to a single-chain antibody made by expressing only the variable region of an antibody through genetic recombination, and refers to a single-chain antibody in which the VH region and VL region of the antibody are connected by a short peptide chain. The term “scFv” is intended to include scFv fragments, including antigen-binding fragments, unless otherwise specified or understood from context. This is self-evident to those skilled in the art.

The epitope, antibody, or antigen-binding fragment thereof according to the present invention may include not only the sequences described herein, but also biological equivalents thereof. For example, additional changes can be made to the amino acid sequence of the antibody to further improve the binding affinity and/or other biological properties of the antibody. Such modifications include, for example, deletions, insertions and/or substitutions of amino acid sequence residues of the antibody. These amino acid mutations are made based on the relative similarity of amino acid side chain substitutions, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape and type of amino acid side chain substitutions shows that arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; Alanine, glycine and serine; And phenylalanine, tryptophan, and tyrosine can be said to be biologically equivalent in function.

In another aspect, the present invention relates to a polynucleotide encoding the amino acid of the antibody or antigen-binding fragment thereof, a recombinant expression vector containing the polynucleotide, and a transformant prepared by co-transforming the vector into a host cell. .

The polynucleotide encoding the antibody or antigen-binding fragment thereof is interpreted to also include sequences showing substantial identity as described above. In the present invention, the polynucleotide may preferably include a base sequence represented by SEQ ID NO: 4, and has at least 60% homology, more specifically 70% homology, with the base sequence represented by SEQ ID NO: 4, More specifically, it may mean a sequence showing 80% homology, most specifically 90% homology.

The polynucleotide is the amino acid sequence of the light and heavy chains of the antibody expressed from the coding region due to codon degeneracy or in consideration of preferred codons in organisms intended to express the light and heavy chains of the antibody or fragments thereof. Various modifications may be made to the coding region within the scope of not changing the coding region, and various modifications or modifications may be made to parts other than the coding region within the scope of not affecting the expression of the gene, and such modified genes are also included in the present invention. Those skilled in the art will be able to understand what is included in the scope. That is, as long as the polynucleotide of the present invention encodes a protein with equivalent activity, one or more nucleic acid bases may be mutated by substitution, deletion, insertion, or a combination thereof, and these are also included within the scope of the present invention. The sequence of these polynucleotides may be single or double stranded and may be DNA molecules or RNA (mRNA) molecules.

When constructing the expression vector, expression control sequences such as promoters, terminators, enhancers, etc., sequences for membrane targeting or secretion, etc. are added depending on the type of host cell in which the antibody is to be produced. It can be selected appropriately and combined in various ways depending on the purpose.

Expression vectors according to the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, etc. A suitable expression vector includes expression control elements such as a promoter, operator, start codon, stop codon, polyadenylation signal, and enhancer, as well as a signal sequence or leader sequence for membrane targeting or secretion, and can be prepared in various ways depending on the purpose. The promoter of an expression vector can be constitutive or inducible. The signal sequence includes the PhoA signal sequence and OmpA signal sequence when the host is Escherichia sp., and the α-amylase signal sequence and subtilisin signal when the host is Bacillus sp. For sequences, etc., if the host is yeast, the MFα signal sequence, SUC2 signal sequence, etc. can be used, and if the host is an animal cell, the insulin signal sequence, α-interferon signal sequence, antibody molecule signal sequence, etc. can be used. It is not limited to this.

Additionally, the expression vector may include a selection marker for selecting host cells containing the vector, and, if it is a replicable expression vector, may include an origin of replication.

The antibody according to the present invention can be mass-produced by transforming the expression vector according to the present invention into an appropriate host cell, such as an E. coli or yeast cell, and then culturing the transformed host cells. Appropriate culture methods and medium conditions depending on the type of host cell can be easily selected by a person skilled in the art from known techniques known to those skilled in the art. The host cell may be a prokaryote such as E. coli or Bacillus subtilis . Additionally, it may be a eukaryotic cell derived from yeast, insect cells, plant cells, or animal cells such as Saccharomyces cerevisiae . More preferably, the animal cells may be autologous or allogeneic animal cells. Transformants produced by introduction into autologous or allogeneic animal cells may be administered to an individual and used in cell therapy to treat cancer. Any method known to those skilled in the art may be used to introduce the expression vector into the host cell.

In another aspect, the present invention relates to a composition for diagnosing cancer, comprising the TMEM165-CLOCK specific antibody or antigen-binding fragment thereof.

Since the TMEM165-CLOCK specific antibody or antigen-binding fragment thereof according to the present invention can specifically bind to TMEM165-CLOCK specifically expressed in cancer cells, it can be used for the purpose of detecting and diagnosing them. That is, the composition contains the TMEM165-CLOCK antibody represented by SEQ ID NO: 3, and the occurrence of cancer can be diagnosed by analyzing the signal shown by the reaction between the sample and the antibody. Specifically, cancer can be diagnosed through the process of confirming the formation of a complex of the TMEM165-CLOCK antibody epitope and a monoclonal antibody or antigen-binding fragment thereof according to the present invention that specifically binds to it, that is, an antigen-antibody complex. . The signal may include, but is not limited to, an enzyme bound to an antibody, such as alkaline phosphatase, β-galactosidase, horse radish peroxidase, luciferase, or cytochrome P450.

The formation of the antigen-antibody complex can be measured according to conventional immunoassay methods, such as ELISA (enzyme linked immunosorbent assay) using an antibody against the TMEM165-CLOCK protein, immunoblotting, western blotting, Detection and measurement may be performed through immunohistochemistry or FACS (Fluorescence-activated cell sorting), but is not limited thereto.

By analyzing the intensity of the final signal from the immunoassay process, the presence or absence of cancer can be diagnosed. In other words, if the TMEM165-CLOCK protein of the present invention is overexpressed in a sample isolated from a suspected individual and the signal is stronger than that of a sample isolated from a normal individual, cancer can be diagnosed.

The cancer may be one or more types selected from the group consisting of stomach cancer, colon cancer, kidney cancer, blood cancer, sarcoma cancer, ovarian cancer, breast cancer, cervical cancer, thyroid cancer, prostate cancer, pancreatic cancer, liver cancer, and lung cancer.

According to one embodiment of the present invention, specific expression of the fusion gene TMEM165-CLOCK was confirmed in various sarcoma cancer tissues such as dermatofibrosarcoma, liposarcoma, undifferentiated pleomorphic sarcoma, and osteochondroma, and in addition to sarcoma cancer, breast cancer, prostate cancer, and lung cancer. As the high expression of TMEM165-CLOCK was confirmed in the cell line, it was confirmed that the fusion gene TMEM165-CLOCK can be presented as a molecular marker for cancer cells. Accordingly, the occurrence of cancer can be diagnosed by measuring the level of formation of the antigen-antibody complex in a sample of a patient suspected of having cancer using the TMEM165_CLOCK specific antibody according to the present invention. As an example, when immunochemical staining (ICC) using a TMEM165_CLOCK-specific antibody was performed to evaluate the TMEM165-CLOCK-specific antibody-antigen affinity for the normal cell line and the sarcoma cancer cell line KHOS/NP, green color was found only in the KHOS/NP cell line. It was confirmed that fluorescent staining was observed, suggesting that the TMEM165-CLOCK specific antibody according to the present invention can detect the cancer cell molecular marker TMEM165_CLOCK, and that sarcoma cancer can be diagnosed through this.

Accordingly, in another aspect, the present invention relates to a method of providing information on cancer diagnosis, comprising reacting the TMEM165-CLOCK specific antibody or antigen-binding fragment thereof with a target sample.

The cancer may be one or more types selected from the group consisting of stomach cancer, colon cancer, kidney cancer, blood cancer, sarcoma cancer, ovarian cancer, breast cancer, cervical cancer, thyroid cancer, prostate cancer, pancreatic cancer, liver cancer, and lung cancer, but is not limited thereto. .

In another aspect, the present invention relates to a composition for preventing or treating cancer, comprising a TMEM165-CLOCK specific antibody or an antigen-binding fragment thereof. The cancer may be one or more types selected from the group consisting of stomach cancer, colon cancer, kidney cancer, blood cancer, sarcoma cancer, ovarian cancer, breast cancer, cervical cancer, thyroid cancer, prostate cancer, pancreatic cancer, liver cancer, and lung cancer, but is not limited thereto. .

The composition according to the present invention may further include PBMC cells or NK cells.

The TMEM165-CLOCK specific antibody or antigen-binding fragment thereof of the present invention can induce more effective cancer cell death by increasing the immunity of immune cells, and can be particularly effectively used in the treatment of cancer in which TMEM165-CLOCK is overexpressed. Therefore, when the TMEM165-CLOCK specific antibody or antigen-binding fragment thereof of the present invention is treated with cancer cells together with an immunotherapeutic agent, such as PBMC cells or NK cells, a more effective cancer prevention or treatment effect can be achieved.

As described above, it was confirmed that TMEM165-CLOCK is overexpressed in cancer cells or cancer tissues, and inhibition of TMEM165-CLOCK using the TMEM165-CLOCK antibody or fragment thereof according to the present invention can be usefully used to inhibit cancer. .

In one embodiment, fluorescence flow cytometry (FACS) using a TMEM165_CLOCK-specific antibody with PBMC cells with increased antibody-specific immunity, and luciferase activity analysis using a TMEM165_CLOCK-specific antibody with NK cells with increased antibody-specific immunity (luciferase activity assay) activity), it was confirmed that cell death increased when sarcoma cell lines harboring TMEM165-CLOCK were treated with TMEM165_CLOCK-specific antibodies.

In one embodiment, it was confirmed that cell proliferation was inhibited when sarcoma cell lines, TMEM165-CLOCK overexpression stabilization cell lines, and lung cancer cell lines were treated with a TMEM165_CLOCK-specific antibody.

In one embodiment, it was confirmed that when a TMEM165_CLOCK-specific antibody was treated with a TMEM165_CLOCK-specific antibody in mice intratibially injected with a stabilized cell line overexpressing TMEM165-CLOCK, the increase in tumor size was suppressed.

According to the above-described embodiment, the TMEM165_CLOCK-specific antibody of the present invention exhibits a therapeutic effect through a TMEM165_CLOCK-specific expression cell line and animal experiments using the same, and the cell proliferation inhibition and killing effects on sarcoma or lung cancer cell lines were confirmed by confirming the TMEM165_CLOCK-specific antibody of the present invention. can show a therapeutic effect if it is a carcinoma with TMEM165_CLOCK overexpression.

The carcinoma may be one or more types selected from the group consisting of stomach cancer, colon cancer, kidney cancer, hematological cancer, sarcoma cancer, ovarian cancer, breast cancer, cervical cancer, thyroid cancer, prostate cancer, pancreatic cancer, liver cancer, and lung cancer.

The composition for preventing or treating cancer may further include pharmaceutically acceptable additives. In this case, the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, and phosphoric acid. Calcium hydrogen, lactose, mannitol, taffy, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, lead carnauba, synthetic aluminum silicate, stearic acid, magnesium stearate, stearic acid. Aluminum, calcium stearate, white sugar, dextrose, sorbitol, and talc can be used. The pharmaceutically acceptable additive according to the present invention is preferably included in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

The composition according to the present invention can be administered in various oral and parenteral formulations during actual clinical administration. When formulated, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants are used. It can be prepared using . Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations include lignified extract and at least one excipient, such as starch, calcium carbonate, and sucrose. It can be prepared by mixing , lactose, or gelatin. Additionally, in addition to simple excipients, lubricants such as magnesium styrate talc may also be used. Liquid preparations for oral use include suspensions, oral solutions, emulsions, and syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. . Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspension solvents include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable esters such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurin, glycerogeratin, etc. can be used.

The composition according to the present invention can be administered orally or parenterally according to the desired method. When administered parenterally, it is administered externally to the skin or intraperitoneally, intrarectally, subcutaneously, intravenously, intramuscularly, or intrathoracically. It is desirable to select the injection method. The dosage range varies depending on the patient's weight, age, gender, health condition, diet, administration time, administration method, excretion rate, and severity of the disease.

The dosage of the composition varies depending on the patient's weight, age, gender, health condition, diet, administration time, administration method, excretion rate, and severity of the disease, and the daily dosage is 0.0001 based on the amount of lignified extract. to 100 mg/kg, preferably 0.001 to 10 mg/kg, and may be administered 1 to 6 times a day.

The antibody according to the present invention can be used alone or in combination with surgery, hormone therapy, drug therapy, and biological response regulators for the treatment of cancer patients. The subject to which the present invention is applicable is a vertebrate animal, preferably a mammal, more preferably an animal such as a rat, rabbit, guinea pig, hamster, dog, or cat, and most preferably an ape animal such as a chimpanzee or gorilla. am.

Hereinafter, the present invention will be described in detail through examples. However, the following examples only specifically illustrate the present invention, and the content of the present invention is not limited by the examples.

Example 1: TMEM165-CLOCK fusion gene screening and expression confirmation

1-1. Sarcoma-specific fusion gene screening

Sarcoma cancer and normal samples were collected from patients who obtained consent for human material research approved by the institutional ethics committee (IRB File No. 2020-02-015-014). Total RNA was extracted from the collected 54 sarcoma and normal tissues, NGS analysis was performed, and sarcoma-specific fusion genes were discovered using the algorithms of the Defuse, FusionCatcher, and Arriba programs.

As shown in Table 1, it was confirmed that among 45 sarcoma tissues, the TMEM165-CLOCK fusion gene was expressed in 9 (20%) sarcoma tissues and was not expressed at all in 9 normal tissues, and TMEM165-CLOCK was found to be expressed in sarcoma tissues. It was predicted that specific new substances (new proteins) would be expressed.

1-2. Verification of sarcoma tissue expression of TMEM165-CLOCK

Based on the above results, it was expected that TMEM165-CLOCK could be used as a marker for diagnosing sarcoma cancer, and PCR was performed on various types of sarcoma tissues to confirm the expression of the TMEM165-CLOCK fusion gene. Forward and reverse primers were prepared (Bionics, Seoul, Korea) to include the fusion junction section of the TMEM165-CLOCK fusion gene and used in the PCR reaction.

PCR primer F: GCTCTTACATTAACATTCT (SEQ ID NO: 5),

PCR primer R:AGTCATCTCAAAGTTCAA (SEQ ID NO: 6)

Figure 1 shows the expression levels of TMEM165-CLOCK in normal cells and various sarcoma tissues. Looking at the top image of Figure 1, when compared to normal tissue (lane 1 and lane 2), the TMEM165-CLOCK fusion gene was significantly expressed in dermatofibrosarcoma protuberans (lane 3) and undifferentiated pleomorphic sarcoma (UPS) tissue in lane 4. You can see that it is happening.

In addition, looking at the bottom image of Figure 1, dermatofibrosarcoma protuberans in lane 1, low-grade liposarcoma in lane 2, undifferentiated pleomorphic sarcoma (UPS) in lane 3, and giant cell tumor (Giant) in lane 4. A high expression level of the TMEM165-CLOCK fusion gene was confirmed in the cell tumor) and osteochondroma tissue in lane 5.

Through the above results, it was confirmed that the TMEM165-CLOCK fusion gene is expressed in various sarcoma cancers such as dermatofibrosarcoma, liposarcoma, undifferentiated pleomorphic sarcoma, and osteochondroma.

1-3. Confirmation of expression of TMEM165-CLOCK in cancer cell lines other than sarcoma

PCR reaction was performed in the same manner as in Example 1-2 to confirm the expression level of the TMEM165-CLOCK fusion gene in various cancer cell lines, including sarcoma.

As shown in Figure 2A, TMEM165-CLOCK in the sarcoma cell line (KHOS/NP, U2OS, MG63 143B) in lanes 1 to 4, the breast cancer cell line (SK-BR3) in lane 5, and the prostate cancer cell line (PC3) in lane 6. It was confirmed that specific expression of the fusion gene occurred.

Additionally, looking at Figure 2b, expression of the TMEM165-CLOCK fusion gene was confirmed in lung cancer cell lines (NCI-H1299, NCI-H1437, NCI-H1793, HCC827, NCI-H23).

Through the above results, it was confirmed that the TMEM165-CLOCK fusion gene is highly expressed in sarcoma cancer, prostate cancer, breast cancer, and lung cancer, and that an agent capable of detecting these genes can diagnose the carcinomas.

Example 2: Synthesis of target antigen peptide

In order to produce an agent capable of detecting TMEM165-CLOCK, a target antigen was prepared. The TMEM165 gene was transcribed in the forward direction (5'→3'), and the CLOCK gene was transcribed in the reverse direction, and the sequences of the combined TMEM165-CLOCK fusion genes were aligned. Looking at Figure 3a, TMEM165 (Homo sapiens It can be seen that it is different from the sequence of transmembrane protein 165; NM_018475.5) (top image). In addition, as shown in Figure 3b, when comparing the amino acid sequence (SEQ ID NO: 3) of TMEM165-CLOCK, it can be seen that amino acids 1 to 299 are identical and amino acids 300 to 321 are different. The sequence from amino acids 300 to 321 with the above differences (SEQ ID NO: 1 below) was determined as the antigen-specific region, and peptide production was performed (Serantec, Korea).

TMEM165 300-321 aa: GSFSPWLILHACSDGKGHLERN (SEQ ID NO: 1)

Based on the sequence analysis results, the peptide sequence 'GSFSPWLILHACSDGKGHLERN (SEQ ID NO: 1)' was synthesized starting from the C-terminus using a solid phase peptide synthesis method. When synthesizing peptides, two types of carrier proteins (BSA, KLH) were conjugated and produced for phage display to obtain the final epitope. Figure 3c shows a schematic diagram of TMEM165-CLOCK and Figure 3d shows the 3D structure of the synthetic peptide.

Example 3: Development of TMEM165-CLOCK specific antibody

3-1. scFv production and selection of anti-TMEM165-CLOCK monoclonal antibody

A phage displaying a single-chain variable fragment (scFv) antibody library was constructed, and biopanning was performed on the epitope synthesized in Example 2. In the case of the phage display library, we selected the human library provided by Bioneer (Korea).

Specifically, the synthesized peptide sequence 'GSFSPWLILHACSDGKGHLERN (SEQ ID NO: 1)' was used as an antigen, and phages from the antibody library were incubated with immobilized targets, and then phages that did not bind to the antigen were filtered out. Once a clone pool binding to the antigen was formed, phages showing high affinity were extracted and single colonies were identified and inoculated respectively. Since the extracted phages were cultured with E. coli host cells for infection and amplification, colonies of infected bacteria were cultured on plates. ELISA was performed on the monoclonal scFv produced in each colony using the above epitope, and positive clones with an OD value of 1.0 or higher were selected.

3-2. Sequence analysis of anti-TMEM165-CLOCK monoclonal antibody

Sequence analysis was performed using the 5'-, 3'-oligonucleotides of the scFv obtained from the Phagemid DNA of the monoclonal antibody prepared from the selected clones. The final sequence of the scFv was identified by combining the common regions of the sequences read in both directions (ABI3730XL, Sanger-method sequencing, Phred Score ≥20), and the TMEM165_CLOCK antibody sequence represented by SEQ ID NO. 2 below was established.

Anti- TMEM165_CLOCK scFv polypeptide (SEQ ID NO: 2): ELELTQPPSVSAAPGQEVTISCSGGNSNLGKNAASWYQQVPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQTGDEADYYCGTWDSRLRGWVFGGGTQLTVLGGGSSRSSSSSGGGGSGGGGQVQLQESGGGGLVQPGRSLRLSCTASGFTFG DYAMSWVRQAPGKGLEWVGFIRSKAYGGTTEYAASVKGRFTISRDDSKSIAYLQMNSLKTEDTAVYYCTRSARRSSLNSGYFDYWGQGTLVTVSSASPTSPKVTSG

Example 4: Sarcoma cancer diagnosis using TMEM165-CLOCK specific antibody

Immunochemical staining (ICC) using the TMEM165_CLOCK antibody prepared in Example 3 was performed to evaluate TMEM165-CLOCK specific antibody-antigen affinity.

First, ^human umbilical vein endothelial cells HUVEC cells ^2.5 Add 1 μg of TMEM165-CLOCK plasmid DNA and 1 μl of Plus reagent to 75 μl of opti-MEM medium (Gibco), and add 4 μl of Lipofectamine LTX (Invitrogen, USA) to 75 μl of opti-MEM medium (Gibco). After mixing and reacting for 20 minutes, it was treated with KHOS/NP cells and cultured at 37°C incubation, and the medium was replaced after 4 hours. Next, the cells were incubated at 37°C for 48 hours, fixed for 10 minutes at RT using 4% paraformaldehyde, and then washed three times with PBS. Then, the cells were permeabilized for 10 minutes at RT using 0.5% Tx-100 and washed three times with PBS. Next, after blocking with 5% BSA for 30 minutes at RT, the antibody was treated at a concentration of 5 μg/ml, reacted at RT for 3-4 hours, and washed three times with PBS. The secondary antibody (Alexa fluor 488 goat anti-human IgG, Invitrogen, USA) was reacted at a ratio of 1:500 for 1 hour at RT, washed three times with PBS, and mounted by dropping a mounting solution containing DAPI. Next, cells were observed through a confocal microscope.

In Figure 4, the HUVEC cell on the left is a normal cell line without expression of the TMEM165_CLOCK fusion gene, the KHOS/NP cell line in the middle is a sarcoma cancer cell line expressing the TMEM165_CLOCK fusion gene, and the cell line on the right is a cell line that overexpresses the TMEM165_CLOCK fusion gene in the KHOS/NP cell line. am. Looking at this, it can be seen that green fluorescent staining is observed specifically only in KHOS/NP and KHOS/NP_TMEM165_CLOCK cell lines expressing the TMEM165_CLOCK fusion gene, and the TMEM165-CLOCK specific antibody according to the present invention can detect TMEM165_CLOCK, through which It was confirmed that sarcoma cancer can be diagnosed.

Example 5: Inhibition of cancer cells using TMEM165-CLOCK specific antibody

5-1. Sarcoma Cell Death

It was confirmed using fluorescence-activated cell sorting (FACS) that the TMEM165-CLOCK-specific antibody prepared in Example 3 increased the antibody-specific immunity of PBMC cells and specifically destroyed TMEM165_CLOCK-expressing cancer cells. .

Sarcoma cancer cell line KHOS/NP_control_luc (control group) and KHOS/NP_TMEM165-CLOCK_luc (experimental group) cells (EMEM medium, hyclone) overexpressing the TMEM165_CLOCK fusion gene in the sarcoma cancer cell line were incubated with CFSE (dojindo, Japan) was dyed at 37°C for 20 minutes. Control group KHOS/NP_control_luc and experimental group KHOS/ ^NP_TMEM165 -CLOCK_luc cells were inoculated into 3 Dishes treated with a concentration of 0.1 ㎍/㎖ and dishes not treated with TMEM165-CLOCK specific antibody were cultured overnight at 37°C.

Then, cells were harvested and stained with FVD (Fixable Viability Dye eFluorTM 780, Invitrogen, USA) for 20 minutes at 4°C, washed twice with PBS containing 2% FBS, and cell death was measured using FACS.

The middle image of Figure 5A (Ab_0 ㎍/㎖) is when no antibody was added, and 7.4% cell death was observed at this time, but the right image (Ab_0.1 ㎍/㎖) is when 0.1 ㎍/㎖ of antibody was treated. Looking at , it can be seen that cell death increases to 45.5%.

This is a TMEM165-CLOCK specific antibody This result shows that cancer cell killing activity against TMEM165_CLOCK-expressing cancer cells can be increased by increasing the antibody-specific immunity of PBMC cells.

Whether the TMEM165-CLOCK specific antibody prepared in Example 3 could increase the antibody-specific immunity of NK cells was confirmed, and cell viability was confirmed through luciferase activity.

KHOS/NP_TMEM165-CLOCK_luc cells, which overexpressed the TMEM165_CLOCK fusion gene in a sarcoma cell line, were inoculated at 1 x 10 ⁴ cells/96 well plate. Next, NK-92 cells (MyeloCult H5100 mdeia, Stemcell Technologies, Canada) were co-cultured (inoculated) at a 1:1 ratio and treated with TMEM165-CLOCK specific antibody at a concentration of 5 ㎍/㎖ in a 96 well plate and TMEM165-CLOCK specific antibody. A 96 well plate without antibody treatment was incubated overnight at 37°C. Then, cells were harvested and luciferase activity was measured (promega, USA).

Looking at the cancer cell survival rate in Figure 5b, in the case of KHOS/NP_TMEM165-CLOCK_luc overexpressing TMEM165-CLOCK, compared to Ab._0 that was not treated with TMEM165-CLOCK specific antibody, it was treated with TMEM165-CLOCK specific antibody at a concentration of 5 μg/ml. In Ab._5, it was confirmed that there was a 20% decrease in cell viability due to the antibody.

5-2. Inhibits sarcoma and lung cancer cell proliferation

Sarcoma cell line (KHOS/NP), TMEM165-CLOCK overexpression stabilization cell line (KHOS/NP_TMEM165-CLOCK), and lung cancer cell line (NCI-H1793) were seeded at 3 x 10 ³ cells/96 well, and after 24 hours, TMEM165-CLOCK MTS assay was performed by treating specific antibodies at a concentration of 50 μg/ml.

Looking at Figure 6 a to c, sarcoma cell line (KHOS/NP), TMEM165-CLOCK overexpression stabilization cell line (KHOS/NP_TMEM165-CLOCK), and lung cancer cell line (NCI-H1793) all showed cell proliferation by treatment with TMEM165-CLOCK specific antibody. It can be seen that (cell proliferation) was significantly reduced.

Through Example 5, the TMEM165-CLOCK specific antibody according to the present invention not only inhibits proliferation and induces death of sarcoma and lung cancer cells, but also inhibits the growth of TMEM165-CLOCK overexpressing cells, thereby treating cancer cells with TMEM165-CLOCK overexpression. It was confirmed that it is useful.

Example 6: Tumor inhibition using TMEM165-CLOCK specific antibody

The antitumor effect of the TMEM165-CLOCK specific antibody prepared in Example 3 was investigated in vivo.

The TMEM165-CLOCK overexpressing stable cell line was injected into the tibia bone at 1 x 10 ⁵ cells/mouse in 6-week-old balb/c-nude mice. The control group (Control) was injected with 1 The sizes of tumors were compared.

Looking at Figure 7 a and b, it was confirmed that the control group (Control) showed a significantly high time-dependent increase in tumor size, whereas the experimental group (TMEM165-CLOCK Ab.) showed a low increase in tumor size.

Example 6 demonstrated that the TMEM165-CLOCK specific antibody according to the present invention exhibits a direct antitumor effect on carcinomas with TMEM165-CLOCK overexpression.

Claims (17)

delete delete delete delete An antibody or antigen-binding fragment thereof consisting of the amino acid sequence shown in SEQ ID NO: 2. The antibody or antigen-binding fragment thereof according to claim 5, wherein the antibody is a single chain antibody (scFv). delete A polynucleotide encoding the antibody or antigen-binding fragment thereof of claim 5. A recombinant expression vector comprising the polynucleotide of claim 8. A transformant other than a human transformed with the vector of claim 9. A composition for diagnosing cancer, comprising the antibody of claim 5 or an antigen-binding fragment thereof. The method of claim 11, wherein the cancer is at least one selected from the group consisting of stomach cancer, colon cancer, kidney cancer, blood cancer, sarcoma cancer, ovarian cancer, breast cancer, cervical cancer, thyroid cancer, prostate cancer, pancreatic cancer, liver cancer, and lung cancer. A composition for diagnosing cancer. A pharmaceutical composition for preventing or treating cancer, comprising the antibody or antigen-binding fragment thereof of claim 5. The method of claim 13, wherein the cancer is one or more types selected from the group consisting of stomach cancer, colon cancer, kidney cancer, blood cancer, sarcoma cancer, ovarian cancer, breast cancer, cervical cancer, thyroid cancer, prostate cancer, pancreatic cancer, liver cancer, and lung cancer. A pharmaceutical composition for preventing or treating cancer. The pharmaceutical composition for preventing or treating cancer according to claim 13, wherein the composition further comprises PBMC cells or NK cells. A method of providing information on cancer diagnosis, comprising reacting the antibody or antigen-binding fragment thereof of claim 5 with a target sample. The method of claim 16, wherein the cancer is one or more types selected from the group consisting of stomach cancer, colon cancer, kidney cancer, blood cancer, sarcoma cancer, ovarian cancer, breast cancer, cervical cancer, thyroid cancer, prostate cancer, pancreatic cancer, liver cancer, and lung cancer. A method of providing information about a cancer diagnosis.