KR102589409B1 - Antibodies for binding to g-protein coupled receptor 87 - Google Patents

Abstract

The present invention relates to a novel antibody that binds to human GPR87 and inhibits the signal transduction process. The antibody specific to the extracellular region of GPR87 or a fragment thereof with immunological activity according to the present invention is specific to the N-terminal extracellular region of GPR87. Since it binds to GPR87 and has functional effectiveness in a concentration-dependent manner and inhibits the invasion of lung cancer cells overexpressing GPR87, it can be usefully used for cancer treatment through GPR87 inhibition, and can be used to verify the presence and extent of GPR87 expression. It can also be used in imaging and diagnostic fields.

Description

GPR87 binding antibody {ANTIBODIES FOR BINDING TO G-PROTEIN COUPLED RECEPTOR 87}

The present invention relates to a novel antibody that binds to human GPR87 and inhibits the signal transduction process.

Medicines for cancer treatment are largely divided into small molecule medicines and high molecule medicines. Compared to low molecule medicines, which have relatively greater side effects due to lack of specificity, high molecule medicines with specificity are receiving more attention as therapeutic agents. Cancer cells express immune checkpoint proteins on the cell surface, which are used by normal cells to suppress immune cell activation in order to avoid the killing mechanism of immune cells. Recently, immunotherapy has been used as a method to treat cancer. Research on checkpoint inhibitor proteins is actively underway.

Meanwhile, GPCR (G-protein coupled receptor) is a membrane protein with seven transmembrane regions and includes a broad class of membrane-bound proteins that share various structural and functional properties (Friedricksson et al. Mol Pharmacol. 63(6): 1256-1272, 2003; and Friedricksson et al., Mol. Pharmacol. 67(5): 1414-1425, 2005). GPCR activates G-protein heterotrimers by transmitting signals from outside the cell into the cell, thereby performing essential physiological roles such as response to signaling substances as well as sensation. GPCR not only regulates cell growth, proliferation, migration, and death, but is also directly related to numerous diseases such as cancer and cardiovascular disease, so it is attracting attention as a major drug target material. It accounts for 30 of the new drug target biomaterials currently on the market. It accounts for ~50%.

Antibody drugs have the advantages of high specificity, low side effects, high blood half-life, and defective cell death mechanisms (ADCC, ADCP, CDC) by the Fc region compared to small molecule synthetic drugs, but in the case of GPCR, antigen preparation is very difficult. Because it is difficult and the extracellular region of GPCR that the antibody should target is limited, there are great technical difficulties in developing anti-GPCR antibodies, and the only antibody drug approved for sale targeting CGRPR is Amgen's Aimovig (Erenumab), which targets CGRPR. and Poteligeo (Mogamulizumab) from Kyowa Hakko Kirin, which target CCR4. Therefore, the need to discover new antibodies that specifically bind to GPCR and regulate its activity has emerged.

The purpose of the present invention is to provide an antibody specific for the extracellular region of GPR87 or a fragment thereof with immunological activity.

Additionally, an object of the present invention is to provide an isolated nucleic acid molecule encoding an antibody specific to the extracellular region of GPR87 or a fragment thereof with immunological activity, a vector containing the same, and a host cell transformed with the vector.

Additionally, an object of the present invention is to provide a pharmaceutical composition for treating or preventing cancer.

Additionally, an object of the present invention is to provide a composition for diagnosing cancer.

Additionally, an object of the present invention is to provide a method of providing information for cancer diagnosis.

In addition, the purpose of the present invention is to provide a method for producing an antibody specific for the extracellular region of GPR87 or a fragment thereof with immunological activity.

To achieve the above object, the present invention provides an antibody specific for the extracellular region of GPR87 or a fragment thereof with immunological activity.

Additionally, the present invention provides an isolated nucleic acid molecule encoding an antibody specific to the extracellular region of GPR87 or a fragment having immunological activity thereof, a vector containing the same, and a host cell transformed with the vector.

Additionally, the present invention provides a pharmaceutical composition for treating or preventing cancer.

Additionally, the present invention provides a composition for diagnosing cancer.

Additionally, the present invention provides a method of providing information for cancer diagnosis.

In addition, the present invention provides a method of producing an antibody specific for the extracellular region of GPR87 or a fragment thereof with immunological activity.

The antibody specific for the extracellular region of GPR87 or a fragment thereof with immunological activity according to the present invention specifically binds to the N-terminal extracellular region of GPR87, has functional effectiveness against GPR87 in a concentration-dependent manner, and is effective against lung cancer in which GPR87 is overexpressed. Since cell invasion is suppressed, it can be usefully used for cancer treatment through GPR87 inhibition, and can also be used in imaging and diagnostic fields to verify the presence and extent of GPR87 expression.

Figure 1 is a diagram confirming the purification results of human GPR87 N-terminal peptide antigen protein expressed in animal cells.
Figure 2 is a diagram showing the amino acid sequences of monoclonal antibodies A5 and B1 that specifically bind to the human GPR87 N-terminal peptide antigen.
Figure 3 is a diagram showing the results of analyzing the size and purity of the light and heavy chains of monoclonal antibodies A5 and B1 purified under reducing and non-reducing conditions, respectively.
Figure 4 is a diagram showing the results of antigen-antibody ELISA analysis of the A5 antibody and B1 antibody prepared against the human GPR87 N-terminal peptide antigen.
Figure 5 is a diagram showing the results of analysis of the luminase inhibition activity of the A5 antibody and the B1 antibody using the Tango reporter system.
Figure 6 is a diagram confirming the invasion inhibition efficacy of the produced B1 antibody against lung cancer cell line A547.

Hereinafter, the present invention will be described in detail through embodiments of the present invention with reference to the attached drawings. However, the following embodiments are provided as examples of the present invention, and if it is judged that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the gist of the present invention, the detailed description may be omitted. , the present invention is not limited thereby. The present invention is capable of various modifications and applications within the description of the claims described below and the scope of equivalents interpreted therefrom.

In addition, the terminology used in this specification is a term used to appropriately express preferred embodiments of the present invention, and may vary depending on the intention of the user or operator or the customs of the field to which the present invention belongs. Therefore, definitions of these terms should be made based on the content throughout this specification. Throughout the specification, when a part is said to “include” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.

Throughout this specification, the usual one- and three-letter codes for naturally occurring amino acids are used, as well as generally acceptable codes for other amino acids such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), etc. A three-character code is used. Additionally, amino acids referred to by abbreviations in the present invention are described according to the IUPAC-IUB nomenclature as follows:

Alanine: A, Arginine: R, Asparagine: N, Aspartic Acid: D, Cysteine: C, Glutamic Acid: E, Glutamine: Q, Glycine: G, Histidine: H, Isoleucine: I, Leucine: L, Lysine: K, Methionine : M, phenylalanine: F, proline: P, serine: S, threonine: T, tryptophan: W, tyrosine: Y and valine: V.

In one aspect, the present invention relates to an antibody specific for the extracellular domain of GPR87 (G-protein coupled receptor 87) or a fragment thereof with immunological activity.

In one embodiment, the antibody may be a monoclonal antibody.

In one embodiment, the antibody or immunologically active fragment thereof may specifically bind to an epitope containing the amino acid sequence of SEQ ID NO: 34.

In one embodiment, the antibody specific for the extracellular region of GPR87 of the present invention or a fragment thereof with immunological activity is CDRH (Complementarity determining regions heavy chain) 1 containing the amino acid sequence of SEQ ID NO: 1 or 2, SEQ ID NO: 3, or It may include a VH domain containing CDRH2 containing the amino acid sequence of SEQ ID NO: 4, and CDRH3 containing the amino acid sequence of SEQ ID NO: 5 or 6.

In one embodiment, the antibody or fragment having immunological activity specific to the extracellular region of GPR87 of the present invention includes CDRL1 containing the amino acid sequence of SEQ ID NO: 7, CDRL2 containing the amino acid sequence of SEQ ID NO: 8 or 9, and It may include a VL domain containing CDRL3 containing the amino acid sequence of SEQ ID NO: 10 or 11.

In one embodiment, the antibody specific for the extracellular region of GPR87 of the present invention or the fragment having immunological activity is FR1 comprising the amino acid sequence of SEQ ID NO: 12 or 13, FR2 comprising the amino acid sequence of SEQ ID NO: 14 or 15 , FR3 containing the amino acid sequence of SEQ ID NO: 16 or 17, and FR4 containing the amino acid sequence of SEQ ID NO: 18 or 19.

In one embodiment, the antibody specific for the extracellular region of GPR87 of the present invention or the fragment having immunological activity is FR1 comprising the amino acid sequence of SEQ ID NO: 20 or 21, FR2 comprising the amino acid sequence of SEQ ID NO: 22 or 23 , FR3 containing the amino acid sequence of SEQ ID NO: 24 or 25, and FR4 containing the amino acid sequence of SEQ ID NO: 26 or 27.

In one embodiment, the antibody specific for the extracellular region of GPR87 of the present invention or a fragment thereof with immunological activity has (i) CDRH1 containing the amino acid sequence of SEQ ID NO: 1 or 2, the amino acid sequence of SEQ ID NO: 3 or 4 A VH domain comprising CDRH2 comprising, and CDRH3 comprising the amino acid sequence of SEQ ID NO: 5 or 6; and/or (ii) a VL domain comprising CDRL1 comprising the amino acid sequence of SEQ ID NO: 7, CDRL2 comprising the amino acid sequence of SEQ ID NO: 8 or 9, and CDRL3 comprising the amino acid sequence of SEQ ID NO: 10 or 11. It may include a V domain.

In one embodiment, the antibody specific for the extracellular region of GPR87 of the present invention or a fragment thereof with immunological activity includes i) FR1 comprising the amino acid sequence of SEQ ID NO: 12 or 13, and the amino acid sequence of SEQ ID NO: 1 or 2. CDRH1, FR2 containing the amino acid sequence of SEQ ID NO: 14 or 15, CDRH2 containing the amino acid sequence of SEQ ID NO: 3 or 4, FR3 containing the amino acid sequence of SEQ ID NO: 16 or 17, amino acid sequence of SEQ ID NO: 5 or 6 A VH domain comprising CDRH3 comprising and FR4 comprising the amino acid sequence of SEQ ID NO: 18 or 19; and/or ii) FR1 comprising the amino acid sequence of SEQ ID NO: 20 or 21, CDRL1 comprising the amino acid sequence of SEQ ID NO: 7, FR2 comprising the amino acid sequence of SEQ ID NO: 22 or 23, amino acid sequence of SEQ ID NO: 8 or 9 A VL domain comprising CDRL2 comprising, FR3 comprising the amino acid sequence of SEQ ID NO: 24 or 25, CDRL3 comprising the amino acid sequence of SEQ ID NO: 10 or 11, and FR4 comprising the amino acid sequence of SEQ ID NO: 26 or 27 It may include a V domain containing.

In one embodiment, the antibody specific for the extracellular region of GPR87 of the present invention or a fragment thereof with immunological activity may be an scFv containing the amino acid sequence of SEQ ID NO: 28 or 29.

In one embodiment, the antibody specific for the extracellular region of GPR87 of the present invention or the fragment having immunological activity is CL containing the amino acid sequence of SEQ ID NO: 30, CH1 containing the amino acid sequence of SEQ ID NO: 31, and SEQ ID NO: 32. It may include a CH2 sequence containing the amino acid sequence of or a CH3 sequence containing the amino acid sequence of SEQ ID NO: 33.

In one embodiment, the immunologically active fragment of the present invention is Fab, Fd, Fab', dAb, F(ab'), F(ab') ₂ , scFv (single chain fragment variable), Fv, single chain antibody. , Fv dimers, complementarity-determining region fragments, humanized antibodies, chimeric antibodies, and diabodies, and scFv is more preferable.

In one embodiment, the antibody specific for the extracellular region of GPR87 of the present invention or a fragment thereof with immunological activity can utilize a defective cell death mechanism and induce phagocytosis of leukocytes and CD89-bearing cell lines (antibody-dependent cellular phagocytosis). , ADCP), antibody-mediated cytotoxicity (antibody dependent cellular cytotoxicity, ADCC), or CDC (Complement Dependent Cytotoxicity) can be used.

The antibodies are in whole antibody form as well as functional fragments of the antibody molecule. 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. A functional fragment of an antibody molecule refers to a fragment that possesses an antigen-binding function. Examples of antibody fragments include (i) the variable region (VL) of the light chain, the variable region (VH) of the heavy chain, the constant region (CL) of the light chain, and Fab fragment consisting of the first constant region (CH1) of the heavy chain; (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) a dAb fragment consisting of a VH domain (Ward ES et al., Nature 341:544-546 (1989)); (v) an isolated CDR region; (vi) a bivalent fragment comprising two linked Fab fragments. F(ab')2 fragment; (vii) single chain Fv molecule (scFv) joined by a peptide linker that joins the VH domain and VL domain to form an antigen binding site; (viii) bispecific single chain Fv dimer (PCT/US92/09965) and (ix) diabody WO94/13804, which is a multivalent or multispecific fragment produced by gene fusion.

The antibody or immunologically active fragment thereof of the present invention may be selected from the group consisting of animal-derived antibodies, chimeric antibodies, humanized antibodies, human antibodies, and immunologically active fragments thereof. The antibody may be recombinantly or synthetically produced.

Animal-derived antibodies produced by immunizing an immunized animal with a desired antigen can generally cause immune rejection when administered to humans for therapeutic purposes, and chimeric antibodies have been developed to suppress such immune rejection. A chimeric antibody is one in which the constant region of an animal-derived antibody, which causes an anti-isotype reaction, is replaced with the constant region of a human antibody using genetic engineering methods. Chimeric antibodies have significantly improved anti-isotype responses compared to animal-derived antibodies, but animal-derived amino acids still exist in the variable region, resulting in potential side effects on anti-idiotypic responses. I'm doing it. Humanized antibodies were developed to improve these side effects. It is produced by transplanting the CDR (complementary determining regions) region, which plays an important role in antigen binding, among the variable regions of a chimeric antibody, into the human antibody framework.

The most important thing in CDR grafting technology to produce humanized antibodies is to select an optimized human antibody that can best accept the CDR region of an animal-derived antibody. To this end, use of an antibody database and crystal structure (crystal structure) Structure analysis, molecular modeling technology, etc. are used. However, even when the CDR region of an animal-derived antibody is transplanted onto an optimized human antibody framework, there are cases where amino acids that affect antigen binding are present in the framework of the animal-derived antibody, so antigen-binding ability is not preserved in many cases. Therefore, the application of additional antibody engineering technology to restore antigen binding ability can be said to be essential.

The antibody or fragment thereof with immunological activity may be isolated from a living body (not present in the living body) or non-naturally occurring, for example, synthetically or recombinantly produced. You can.

In the present invention, “antibody” refers to a substance produced by stimulation of an antigen within the immune system, the type of which is not particularly limited, and can be obtained naturally or unnaturally (e.g., synthetically or recombinantly). You can. Antibodies are very stable not only in vitro but also in vivo and have a long half-life, making them advantageous for mass expression and production. In addition, antibodies inherently have a dimer structure, so their adhesion ability (avidity) is very high. A complete 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 constant region of an antibody is divided into a heavy chain constant region and a light chain constant region, and the heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and subclasses. It has gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types.

As used herein, the term "heavy chain" refers to a variable region domain V _H and three constant region domains C _H1 , C _H2 and C _H3 comprising an amino acid sequence with sufficient variable region sequence to confer specificity to an antigen. It is interpreted to include both the full-length heavy chain including the hinge and fragments thereof. Additionally, the term "light chain" refers to both a full-length light chain and fragments thereof comprising a variable region domain V _L and a constant region domain C _L comprising an amino acid sequence with sufficient variable region sequence to confer specificity to an antigen. It is interpreted to mean inclusive.

In the present invention, the term "variable region or variable domain" refers to a portion of an antibody molecule that performs the function of specifically binding to an antigen and exhibits many variations in sequence, and the variable region has complementary There are crystalline regions CDR1, CDR2 and CDR3. A framework region (FR) exists between the CDRs and serves to support the CDR ring. The “complementarity determining region” is a ring-shaped region involved in antigen recognition, and as the sequence of this region changes, the specificity of the antibody to the antigen is determined.

The term "scFv (single chain fragment variable)" used in the present invention refers to a single chain antibody made by expressing only the variable region of an antibody through genetic recombination, and is a single chain form in which the VH region and VL region of the antibody are connected by a short peptide chain. refers to the antibodies of 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.

In the present invention, the term “complementarity determining region (CDR)” refers to the amino acid sequence of the hypervariable region of the heavy and light chains of immunoglobulin. The heavy and light chains may each contain three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). The CDR may provide key contact residues for the antibody to bind to the antigen or epitope.

In the present invention, the term "specifically binds" or "specifically recognizes" has the same meaning commonly known to those skilled in the art, and means that an antigen and an antibody specifically interact to produce an immunological reaction. .

In the present invention, the term “antigen-binding fragment” refers to a fragment of the entire structure of an immunoglobulin and a portion of a polypeptide containing a portion to which an antigen can bind. For example, it may be scFv, (scFv) 2, scFv-Fc, Fab, Fab' or F(ab') 2, but is not limited thereto. Among the antigen-binding fragments, Fab has one antigen-binding site with a structure that includes the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region (C _H1 ) of the heavy chain. Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain C _H1 domain. The F(ab') 2 antibody is produced when the cysteine residue in the hinge region of Fab' forms a disulfide bond. Fv is a minimal antibody fragment containing only the heavy chain variable region and the light chain variable region, and recombinant techniques for producing Fv fragments are widely known in the art. A two-chain Fv (two-chain Fv) is a non-covalent bond in which the heavy chain variable region and a light chain variable region are connected, while a single-chain Fv (single-chain Fv) is generally shared between the heavy chain variable region and the short chain variable region through a peptide linker. They can be connected by a bond or directly connected at the C-terminus to form a dimer-like structure, such as double-chain Fv. The linker may be a peptide linker consisting of 1 to 100 or 2 to 50 amino acids, and suitable sequences are known in the art. The antigen-binding fragment can be obtained using a proteolytic enzyme (for example, Fab can be obtained by restriction digestion of the entire antibody with papain, and F(ab') 2 fragment can be obtained by digestion with pepsin), It can be produced through genetic recombination technology.

In the present invention, the term "hinge region" refers to a region contained in the heavy chain of an antibody, which exists between the C _H1 and C _H2 regions and functions to provide flexibility of the antigen binding site in the antibody. It means area. For example, the hinge may be derived from a human antibody, specifically, IgA, IgE, or IgG, such as IgG1, IgG2, IgG 3, or IgG4.

In one aspect, the present invention relates to an isolated nucleic acid molecule encoding an antibody of the present invention or an immunologically active fragment thereof, a vector containing the same, and a host cell transformed therewith.

Nucleic acid molecules of the invention may be isolated or recombinant and include single- and double-stranded forms of DNA and RNA as well as corresponding complementary sequences. An isolated nucleic acid, in the case of a nucleic acid isolated from a naturally occurring source, is a nucleic acid that has been separated from the surrounding genetic sequence present in the genome of the individual from which the nucleic acid was isolated. In the case of nucleic acids synthesized enzymatically or chemically from a template, such as PCR products, cDNA molecules, or oligonucleotides, the nucleic acids resulting from these procedures may be understood as isolated nucleic acid molecules. Isolated nucleic acid molecules refer to nucleic acid molecules either in the form of separate fragments or as components of larger nucleic acid constructs. A nucleic acid is operably linked when placed in a functional relationship with another nucleic acid sequence. For example, the DNA of the presequence or secretion leader is operably linked to the DNA of the polypeptide when the polypeptide is expressed as a preprotein in a form before secretion, and the promoter or enhancer is a polypeptide sequence. is operably linked to the coding sequence when it affects transcription, or the ribosome binding site is operably linked to the coding sequence when configured to facilitate translation. In general, operably linked means that the DNA sequences to be linked are located adjacent to each other, and in the case of a secretory leader, it means that they are adjacent and exist within the same reading frame. However, enhancers do not need to be located adjacently. Linking is accomplished by ligation at convenient restriction enzyme sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used according to conventional methods.

Isolated nucleic acid molecules encoding the antibodies of the present invention or immunologically active fragments thereof are expressed from the coding region due to codon degeneracy or in consideration of preferred codons in the organism in which the antibody is to be expressed. Various modifications may be made to the coding region as long as they do not change the amino acid sequence of the antibody, and various modifications or modifications may be made to parts other than the coding region as long as they do not affect gene expression, and such modifications may be made to the coding region. Those skilled in the art will understand that genes are also included within the scope of the present invention. That is, as long as the nucleic acid molecule 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 nucleic acid molecules may be single or double stranded, and may be DNA molecules or RNA (mRNA) molecules.

Isolated nucleic acid molecules encoding the antibodies of the present invention or fragments having immunological activity according to the present invention may be inserted into an expression vector for protein expression. Expression vectors typically contain proteins operably linked, i.e., placed in a functional relationship, with regulatory or control sequences, selectable markers, optional fusion partners, and/or additional elements. Under appropriate conditions, a host cell transformed with a nucleic acid, preferably containing an antibody of the invention or an immunologically active fragment thereof, or an isolated nucleic acid molecule encoding a bispecific or multispecific antibody of the invention, is expressed. An antibody of the present invention, a fragment having immunological activity thereof, or a bispecific or multispecific antibody of the present invention can be produced by culturing a vector to induce protein expression. A variety of suitable host cells can be used, including, but not limited to, mammalian cells, bacteria, insect cells, and yeast. Methods for introducing exogenous nucleic acids into host cells are known in the art and will vary depending on the host cell used. Preferably, E. coli, which has low production cost and thus has high industrial value, can be produced as a host cell.

Vectors of the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, etc. Suitable vectors include expression control elements such as promoters, operators, start codons, stop codons, polyadenylation signals, and enhancers, as well as signal sequences or leader sequences for membrane targeting or secretion, and can be prepared in various ways depending on the purpose. The promoter of the vector may 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 vector may include a selection marker for selecting host cells containing the vector, and if it is a replicable expression vector, it will include an origin of replication.

In the present invention, the term “vector” refers to a carrier capable of inserting a nucleic acid sequence for introduction into a cell capable of replicating the nucleic acid sequence. Nucleic acid sequences may be exogenous or heterologous. Vectors include, but are not limited to, plasmids, cosmids, and viruses (eg, bacteriophages). Those skilled in the art can construct vectors by standard recombination techniques (Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1988; and Ausubel et al., In: Current Protocols in Molecular Biology, John, Wiley & Sons, Inc, NY, 1994, etc.).

In one embodiment, when constructing the vector, expression control sequences such as promoters, terminators, enhancers, etc., depending on the type of host cell for producing the antibody, membrane targeting or secretion Sequences, etc. can be appropriately selected and combined in various ways depending on the purpose.

In the present invention, the term “expression vector” refers to a vector containing a nucleic acid sequence encoding at least a portion of the gene product to be transcribed. In some cases, the RNA molecule is then translated into a protein, polypeptide, or peptide. Expression vectors may contain various control sequences. In addition to regulatory sequences that regulate transcription and translation, vectors and expression vectors may also contain nucleic acid sequences that also serve other functions.

In the present invention, the term “host cell” includes eukaryotes and prokaryotes and refers to any transformable organism capable of replicating the vector or expressing the gene encoded by the vector. The host cell may be transfected or transformed by the vector, which refers to a process in which an exogenous nucleic acid molecule is transferred or introduced into the host cell.

In one embodiment, the host cells may be bacteria or animal cells, the animal cell line may be CHO cells, HEK cells, or NSO cells, and the bacteria may be Escherichia coli.

In one aspect, the present invention relates to a GPR87 inhibitor comprising the antibody of the present invention or a fragment thereof with immunological activity.

In one aspect, the present invention relates to a pharmaceutical composition for the treatment or prevention of a GPR87 overexpression disease comprising the antibody of the present invention or an immunologically active fragment thereof as an active ingredient.

In one aspect, the present invention relates to a pharmaceutical composition for the treatment or prevention of cancer comprising the antibody of the present invention or an immunologically active fragment thereof as an active ingredient.

In one embodiment, the cancer is brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, and colon cancer. , small intestine cancer, rectal cancer, fallopian tube carcinoma, anal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymph node cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer. , soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, renal or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma. and pituitary adenoma, and more preferably pancreatic cancer, uterine cancer, or lung cancer.

In one embodiment, the composition of the present invention can inhibit cancer invasion or metastasis.

The pharmaceutical composition of the present invention can be used as a stand-alone therapy, but can also be used in combination with other conventional biological therapies, chemotherapy, or radiotherapy. When such combination therapy is performed, cancer can be treated more effectively. When the present invention is used for the prevention and treatment of cancer, chemotherapy agents that can be used with the composition include cisplatin, carboplatin, procarbazine, mechlorethamine, Cyclophosphamide, ifosfamide, melphalan, chlorambucil, bisulfan, nitrosourea, dactinomycin, daunoru daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin, etoposide, tamoxifen, taxol, transpla Includes transplatinum, 5-fluorouracil, vincristin, vinblastin and methotrexate. Radiation therapy that can be used with the composition of the present invention includes X-ray irradiation and γ-ray irradiation.

In the present invention, the term “prevention” refers to all actions that inhibit or delay the occurrence, spread, and recurrence of cancer by administering the PD-1 variant or a composition containing the same according to the present invention.

The therapeutically effective amount of the composition of the present invention may vary depending on various factors, such as administration method, target site, patient condition, etc. Therefore, when used in the human body, the dosage must be determined as appropriate by considering both safety and efficiency. It is also possible to estimate the amount used in humans from the effective amount determined through animal testing. These considerations in determining an effective amount include, for example, Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; and E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used in the present invention, the term "pharmaceutically effective amount" refers to an amount that is sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment and does not cause side effects, and the effective dose level is determined by the patient's Factors including health status, type and severity of disease, activity of drug, sensitivity to drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, drugs combined or used simultaneously, and other factors well known in the field of medicine. It can be decided depending on The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.

The pharmaceutical composition of the present invention may contain a carrier, diluent, excipient, or a combination of two or more commonly used in biological products. As used in the present invention, the term “pharmaceutically acceptable” means that the composition exhibits non-toxic properties to normal cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. The compounds described in, saline solution, sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these ingredients can be mixed and used, and if necessary, other ingredients such as antioxidants, buffers, and bacteriostatic agents. Normal additives can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate dosage forms such as aqueous solutions, suspensions, emulsions, etc., into pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group including oral formulations, topical formulations, suppositories, sterile injectable solutions, and sprays.

The composition of the present invention may also include carriers, diluents, excipients, or combinations of two or more commonly used in biological products. Pharmaceutically acceptable carriers are not particularly limited as long as they are suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. The compounds described in, saline solution, sterilized water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these ingredients can be mixed and used, and if necessary, other ingredients such as antioxidants, buffers, and bacteriostatic agents. Normal additives can be added. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate dosage forms such as aqueous solutions, suspensions, emulsions, etc., into pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the art or a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

The composition of the present invention may additionally contain one or more active ingredients that exhibit the same or similar functions. The composition of the present invention contains 0.0001 to 10% by weight of the protein, preferably 0.001 to 1% by weight, based on the total weight of the composition.

The pharmaceutical composition of the present invention may further include pharmaceutically acceptable additives, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, and calcium hydrogen phosphate. , 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, aluminum stearate, Calcium stearate, white sugar, dextrose, sorbitol, and talc may be used. The pharmaceutically acceptable additive according to the present invention is preferably contained in an amount of 0.1 to 90 parts by weight based on the composition, but is not limited thereto.

The composition of the present invention can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) or orally depending on the desired method, and the dosage is determined by the patient's weight, age, gender, health condition, The range varies depending on diet, administration time, administration method, excretion rate, and severity of disease. The daily dosage of the composition according to the present invention is 0.0001 to 10 mg/ml, preferably 0.0001 to 5 mg/ml, and it is more preferable to administer it once or several times a day.

Liquid preparations for oral administration of the composition of the present invention include suspensions, oral solutions, emulsions, syrups, etc., and in addition to the commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives are used. etc. may be included together. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, suppositories, etc.

In one aspect, the present invention relates to a composition for diagnosing GPR87 overexpression disease comprising an antibody specific for the extracellular region of GPR87 or a fragment thereof with immunological activity.

In one aspect, the present invention relates to a composition for cancer diagnosis comprising an antibody specific for the extracellular region of GPR87 or a fragment thereof with immunological activity.

As used herein, the term “detection” or “measurement” means quantifying the concentration of a detected or measured object.

In one aspect, the present invention relates to a cancer diagnostic kit comprising the composition for cancer diagnosis of the present invention.

In one embodiment, the kit may further include tools and/or reagents for collecting a biological sample from a subject or patient, as well as tools and/or reagents for preparing genomic DNA, cDNA, RNA or protein from the sample. there is.

In the present invention, the term “cancer diagnostic kit” refers to a kit containing the cancer diagnostic composition of the present invention. Therefore, the above expression “cancer diagnostic kit” can be used interchangeably or interchangeably with “cancer diagnostic composition.” As used herein, the term “diagnosis” refers to determining the susceptibility of an object to a specific disease or disorder, determining whether an object currently has a specific disease or disorder, and determining whether an object currently has a specific disease or disorder. Determining a subject's prognosis (e.g., identifying a pre-metastatic or metastatic cancer state, determining the stage of the cancer, or determining the responsiveness of the cancer to treatment), or therametrics (e.g., determining the efficacy of a treatment) Includes monitoring the status of an object to provide information about it.

The antibody specific to the extracellular region of GPR87 of the present invention or its fragment with immunological activity is specific to GPR87, which is overexpressed in various cancers and directly or indirectly participates in the progression of cancer, playing a critical role in the survival rate and prognosis of cancer patients. Since it binds aggressively, it can be used as a theranostic agent also used in the diagnosis of cancer. In addition, CAR (chimeric antigen receptor)-T cells containing scFv can be produced and used as an anticancer agent, and can also be used as an anticancer adjuvant administered simultaneously, separately, or sequentially along with an anticancer agent. In addition, it can also be used as a drug delivery vehicle to target cancer cells.

In one aspect, the present invention provides the following steps: reacting a biological sample isolated from a subject with an antibody specific for the extracellular region of GPR87 of the present invention or a fragment thereof with immunological activity; Confirming the expression level of GPR87; and comparing the expression level of GPR87 in a normal control sample.

In one embodiment, when the expression level of GPR87 in the biological sample isolated from the subject is higher than the expression level of GPR87 in the normal control sample, a step of determining that the test subject has cancer may be further included.

As used herein, the term “sample” refers to a biological sample obtained from a subject or patient. Sources of biological samples include solid tissue from fresh, frozen and/or preserved organ or tissue samples or biopsies or aspirates; Blood or any blood component; It may be a cell from any point in the subject's pregnancy or development.

In one aspect, the present invention provides the steps of culturing a host cell transformed with a vector containing an isolated nucleic acid molecule encoding an antibody of the present invention or a fragment having immunological activity thereof; and recovering the antibody or immunologically active fragment thereof from the host cell culture.

The present invention will be described in more detail through the following examples. However, the following examples are only for illustrating the content of the present invention and are not intended to limit the present invention.

Example 1. Expression and purification of N-terminal extracellular antigen of human GPR87

The sequence of the N-terminal extracellular region of human GPR87 required for antibody discovery was referenced to (Uniprot ID: Q9BY21), and for efficient antigen production, the human GPR87 N-terminal extracellular region (SEQ ID NO: 34) and GST ( Glutathione s transferase) and STV (Streptavidin) fused gene sequences (SEQ ID NOs: 35 and 36, respectively) (Table 1) were inserted into pMAZ, an expression vector for animal cells, to complete the expression vector. This was transfected into animal cells Expi293 and cultured in Freestyle293 expression medium (Life Technologies, USA) at 37°C for 7 days. The expressed cell culture was centrifuged at 6,000 rpm for 20 minutes, and the supernatant was taken and filtered through a 0.22 μm filter. The filtered supernatant expressing the GST-fused antigen was mixed with 1 ml of GST agarose (Genscript, USA) resin, and the supernatant expressing the STV-fused antigen was mixed with 1 ml of Ni-NTA agarose (Qiagen, USA) resin at 4°C. Binding was induced for 16 hours. The combined resin was washed with 10 CV (column volume) of PBS solution or PBS containing 20mM Imidazole, then washed with PBS containing 10mM reduced glutathione or PBS containing 250mM Imidazole. After elution, the buffer was changed to PBS to produce human GPR87 N-terminal extracellular antigen, and the size and purity of the two purified antigens were analyzed by SDS-PAGE (Figure 1).

order sequence number N-terminal extracellular region of human GPR87 MGFNLTLAKLPNNELHGQESHNSGNRSDGPGKNTTLHNEFD 34 GPR87(N)-GST MGFNLTLAKLPNNELHGQESHNSGNRSDGPGKNTTLHNEFDMSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLY MDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPKRS 35 GPR87(N)-Streptavidin MGFNLTLAKLPNNELHGQESHNSGNRSDGPGKNTTLHNEFDDPSKESKAQAAVAEAGITGTWYNQLGSTFIVTANPDGSLTGTYESAVGNAESRYVLTGRYDSTPATDGSGTALGWTVAWKNNYRNAHSATTWSGQYVGGAEARINTQWLLTSGTTAANAWKSTLVGHDTFTKVKPSAASIDAAKKAGVNNGNPLDAVQQGSHHHHHH 36

Example 2. Discovery of antibodies specific to the N-terminal extracellular region of human GPR87

To remove clones that bind to GST or STV fused to the N-terminal extracellular region peptide of human GPR87 among the phage libraries on which human single-chain antibodies are surface-expressed, purified phage immobilized on an ELISA plate (Coning, USA) Negative sorting was performed for each round of biopanning by reacting 10 ^{to 12} phage libraries with GST or STV protein at room temperature and then taking the supernatant. Biopanning was performed by reacting the supernatant after negative separation on an ELISA plate immobilized with a protein antigen fused to GPR87 N-terminal peptide with GST or STV at room temperature. After reacting at room temperature for 1 hour, the supernatant was removed, washed with PBST (PBS containing 0.05% Tween-20), and bound phages were eluted with a low pH elution buffer (glycine-HCl pH 2.2). was neutered. The eluted phage was amplified using VCSM13 helper phage and ER2738 E. coli, and the amplified phage was repeatedly subjected to the biopanning described above by increasing the number of washes each time. Biopanning was performed a total of four times through the above process, and the titer results for each round are shown in Table 2. After performing individual phage ELISA on proteins fused to the human GPR87 N-terminus with GST or STV on 200 clones among the eluted phages after four rounds of biopanning, 10 types showing the highest binding affinity to the human GPR87 N-terminal antigen were identified. By sequencing the clones, the final two types of monoclonal antibodies (A5 and B1) were obtained and their sequences were confirmed (Figure 2 and Table 3).

order sequence number VH CDRH1 A5 GGSMTSYY One B1 GFNFDEYA 2 CDRH2 A5 VFHSGIT 3 B1 IRSKAYRETT 4 CDRH3 A5 AKKSGTSKKPIDY 5 B1 ARGHTPQRPYFDE 6 V.L. CDRL1 A5, B1 QSVGTY 7 CDRL2 A5 DAS 8 B1 TAS 9 CDRL3 A5 LQHKNYPLT 10 B1 QQSYRPHFLT 11 VH FR1 A5 QVQLVQSGPGLVKPSETLSLTCSVS 12 B1 EVQLVESGGGLVQPGRSLRLSCKAS 13 FR2 A5 WSWVRQPPGKGLEWMGY 14 B1 LSWVRQAPGKGLEWVGF 15 FR3 A5 NYSPSLKSRVTMSVDTSKNQFSLKLKSVTAVDTAVYYC 16 B1 EYAASVKGRCTISRDDSKSIAYLLMNSLRAEDTAVYYC 17 FR4 A5 WGQGTTVTVSS 18 B1 WGQGTLVTVSS 19 V.L. FR1 A5 DIVMTQSPSSLSASVGDRITITCRAS 20 B1 DIQMTQSPSFLSASVGDRITITCRAS 21 FR2 A5 VNWYQHKPGKAPKLLIY 22 B1 VNWYQQKLGKAPKLLIY 23 FR3 A5 NLETGVPSRFSGSGSVTDFTLTITNLQPEDSATYYC 24 B1 TLQSGVPARFSGSGSGTEFTLTINGLQPEDFATYYC 25 FR4 A5 FGGGTKVEIKR 26 B1 FGGGTQLTVLG 27 A5 (scFv) QVQLVQSGPGLVKPSETLSLTCSVSGGSMTSYYWSWVRQPPGKGLEWMGYVFHSGITNYSPSLKSRVTMSVDTSKNQFSLKLKSVTAVDTAVYYCAKKSGTSKKPIDYWGQGTTVTVSSGGGSSGGGGSGGGGSGGGGSDIVMTQSPSSLSASVGDRITITCRASQSVGTYVNWYQHKPGKAPKLLIYDASNLETG VPSRFSGSGSVTDFTLTITNLQPEDSATYYCLQHKNYPLTFGGGTKVEIKR 28 B1 (scFv) EVQLVESGGGLVQPGRSLRLSCKASGFNFDEYALSWVRQAPGKGLEWVGFIRSKAYRETTEYAASVKGRCTISRDDSKSIAYLLMNSLRAEDTAVYYCARGHTPQRPYFDEWGQGTLVTVSSGGGSSGGGGSGGGGSGGGGSDIQMTQSPSFLSASVGDRITITCRASQSVGTYVNWYQQKLGKAPKLLIYTASTLQSG VPARFSGSGSGTEFTLTINGLQPEDFATYYCQQSYRPHFLTFGGGTQLTVLG 29 CL TVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 30 CH1 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV 31 CH2
EPPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK 32 CH3 GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 33

Example 3. Expression and purification of two antibodies binding to human GPR87 N-terminus

An expression vector was prepared by converting the two monoclonal antibodies obtained into immunoglobulin-type IgG, and the recovered expression vector was amplified through E. coli transformation in a 150 mM NaCl solution containing PEI (polyethylenimine, Polysciences, USA). Transfection was used to transiently express expression in Expi293 cells and cultured in Freestyle293 expression medium (Life Technologies, USA) at a temperature of 37°C for 7 days. The expressed cell culture was centrifuged at 6,000 rpm for 20 minutes, and the supernatant was taken and filtered through a 0.22 μm filter. The filtered supernatant was induced to bind to 1 ml of protein A agaroe (Amicogen, Korea) resin for 16 hours at 4°C. The combined resin was washed with 10 CV of PBS solution, eluted with 100 mM glycine (pH 2.7) solution, and neutralized with 1 M Tris-HCl (pH 8.0). After changing the buffer to PBS, the size and purity of the light and heavy chains of the two purified monoclonal antibodies (A5 and B1) were analyzed by SDS-PAGE under reducing and non-reducing conditions, respectively (Figure 3).

Example 4. Analysis of binding ability of anti-GPR87 antibody to antigen

The GPR87 N-terminal antigen binding ability of the two monoclonal antibodies (A5 and B1) prepared in the above example was analyzed by ELISA. Specifically, antigens fused to human GPR87 N-terminal extracellular region peptide with GST or STV were diluted with 0.05 M Na ₂ CO ₃ (pH 9.6) buffer solution and incubated at a concentration of 4 μg/well on ELISA plates (Corning, USA). ) at 4°C for 16 hours, and blocked at room temperature for 1 hour with a PBS solution containing 4% skim milk. After removing the skim milk, the two monoclonal antibodies (A5 and B1) expressed and purified in the above example were serially diluted 4-fold and reacted at 50 μl/well on an ELISA plate for 1 hour at room temperature. After washing four times with 150 μl/well of PBST solution, human IgG(H+L)-HRP conjugate was diluted with PBS at a ratio of 1:5,000 and reacted at room temperature for 1 hour in each ELISA plate. I ordered it. After removing the solution, the solution was washed four times with 150 μl/well of PBST solution and reacted with 50 μl/well of TMB (Thermo Scientific). After 20 minutes, the reaction was terminated with 4 NH ₂ SO ₄ and the absorbance was measured at 450 nm. did.

As a result of ELISA signal analysis, the binding affinity of the control Herceptin antibody was very low and no visible equilibrium dissociation constant was measured, while the visible equilibrium dissociation constants of A5 were 13.34 nM for GPR87-GST and 14.67 nM for GPR87-STV. It was found that the visible equilibrium dissociation constant of B1 was 18.06 nM for GPR87-GST and 19.85 nM for GPR87-STV (Figure 4 and Table 4).

Example 5. Anti-GPR87 antibody in vitro Functional validation

The in vitro anticancer effectiveness of the discovered antibody was verified using the Tango reporter system, which exhibits luciferase inhibitory activity by an antagonist acting on a specific GPCR (Nat Struct Mol Biol. 2015 May;22(5) :362-9). Specifically, the GPR87 expression vector was transfected into the HTLA cell line expressing the Tango reporter gene, and 6 hours later, the cells were replaced with culture medium and cultured. After 24 hours, the two types of monoclonal antibodies of the present invention (A5 and B1) were treated at different concentrations, and after 4 hours, they were treated with LPA (Lysophosphatidic acid), an agonist for GPR87, and cultured. After 48 hours, in vitro functional effectiveness was verified using a Dual luciferase reporter assay kit (Promega, USA).

As a result, the monoclonal antibodies A5 and B1 of the present invention each showed the effect of effectively inhibiting the activity of the luminescent enzyme depending on the concentration (FIG. 5). Through the above results, it was confirmed that the monoclonal antibodies A5 and B1 of the present invention have in vitro functional effectiveness.

Example 6. Verification of the efficacy of anti-GPR87 antibody to inhibit cancer cell invasion

In order to confirm the effect of the anti-GPR87 antibody of the present invention on lung cancer disease, which is known to be one of the indications associated with GPR87 overexpression, the lung cancer cell line A549 cell line was treated with the monoclonal antibody B1 of the present invention at a concentration of 100 μg and Transwell inserted. were inoculated and cultured for 24 hours. After 24 hours of culture, the infiltrated cells were stained using Diff Quic dye (Sysmex, Japan) to confirm the effect of inhibiting the invasion of lung cancer cell lines.

As a result, while the control group, Human IgG, did not show an invasion inhibitory effect, the monoclonal antibody B1 of the present invention was shown to effectively inhibit the invasion of the lung cancer cell line A549 (FIG. 6). Through this, it was confirmed that the anti-GPR87 monoclonal antibody of the present invention has the effect of effectively inhibiting the invasion of lung cancer cells.

<110> Korea University Research and Business Foundation <120> ANTIBODIES FOR BINDING TO G-PROTEIN COUPLED RECEPTOR 87 <130> DP-2020-0804 <160> 36 <170> KoPatentIn 3.0 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> A5-CDRH1 <400> 1 Gly Gly Ser Met Thr Ser Tyr Tyr 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> < 223> B1-CDRH1 <400> 2 Gly Phe Asn Phe Asp Glu Tyr Ala 1 5 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> A5-CDRH2 <400> 3 Val Phe His Ser Gly Ile Thr 1 5 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> B1-CDRH2 <400> 4 Ile Arg Ser Lys Ala Tyr Arg Glu Thr Thr 1 5 10 <210> 5 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> A5-CDRH3 <400> 5 Ala Lys Lys Ser Gly Thr Ser Lys Lys Pro Ile Asp Tyr 1 5 10 <210> 6 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> B1-CDRH3 <400> 6 Ala Arg Gly His Thr Pro Gln Arg Pro Tyr Phe Asp Glu 1 5 10 <210> 7 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> A5/B1-CDRL1 <400> 7 Gln Ser Val Gly Thr Tyr 1 5 <210> 8 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> A5-CDRL2 <400> 8 Asp Ala Ser 1 <210> 9 <211> 3 <212> PRT <213> Artificial Sequence <220> <223> B1-CDRL2 <400> 9 Thr Ala Ser 1 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> A5-CDRL3 <400> 10 Leu Gln His Lys Asn Tyr Pro Leu Thr 1 5 <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> B1-CDRL3 <400> 11 Gln Gln Ser Tyr Arg Pro His Phe Leu Thr 1 5 10 <210> 12 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> A5-VH-FR1 <400> 12 Gln Val Gln Leu Val Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val Ser 20 25 <210 > 13 <211> 25 <212> PRT <213> Artificial Sequence <220> <223> B1-VH-FR1 <400> 13 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Lys Ala Ser 20 25 <210> 14 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> A5-VH-FR2 <400> 14 Trp Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met Gly 1 5 10 15 Tyr <210> 15 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> B1-VH-FR2 <400> 15 Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly 1 5 10 15 Phe <210> 16 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> A5-VH-FR3 <400> 16 Asn Tyr Ser Pro Ser Leu Lys Ser Arg Val Thr Met Ser Val Asp Thr 1 5 10 15 Ser Lys Asn Gln Phe Ser Leu Lys Leu Lys Ser Val Thr Ala Val Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 17 < 211> 38 <212> PRT <213> Artificial Sequence <220> <223> B1-VH-FR3 <400> 17 Glu Tyr Ala Ala Ser Val Lys Gly Arg Cys Thr Ile Ser Arg Asp Asp 1 5 10 15 Ser Lys Ser Ile Ala Tyr Leu Leu Met Asn Ser Leu Arg Ala Glu Asp 20 25 30 Thr Ala Val Tyr Tyr Cys 35 <210> 18 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> A5-VH-FR4 <400> 18 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 1 5 10 <210> 19 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> B1 -VH-FR4 <400> 19 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 20 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> A5-VL-FR1 <400> 20 Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Ile Thr Ile Thr Cys Arg Ala Ser 20 25 <210> 21 <211> 26 <212> PRT <213 > Artificial Sequence <220> <223> B1-VL-FR1 <400> 21 Asp Ile Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Ile Thr Ile Thr Cys Arg Ala Ser 20 25 <210> 22 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> A5-VL-FR2 <400> 22 Val Asn Trp Tyr Gln His Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 23 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> B1-VL-FR2 <400> 23 Val Asn Trp Tyr Gln Gln Lys Leu Gly Lys Ala Pro Lys Leu Leu Ile 1 5 10 15 Tyr <210> 24 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> A5-VL-FR3 <400> 24 Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Val 1 5 10 15 Thr Asp Phe Thr Leu Thr Ile Thr Asn Leu Gln Pro Glu Asp Ser Ala 20 25 30 Thr Tyr Tyr Cys 35 <210> 25 <211> 36 <212> PRT <213> Artificial Sequence < 220> <223> B1-VL-FR3 <400> 25 Thr Leu Gln Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr Glu Phe Thr Leu Thr Ile Asn Gly Leu Gln Pro Glu Asp Phe Ala 20 25 30 Thr Tyr Tyr Cys 35 <210> 26 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> A5-VL-FR4 <400> 26 Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 1 5 10 <210> 27 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> B1-VL-FR4 <400> 27 Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly 1 5 10 <210> 28 <211> 247 <212> PRT <213> Artificial Sequence <220> <223> A5 <400> 28 Gln Val Gln Leu Val Gln Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val Ser Gly Gly Ser Met Thr Ser Tyr 20 25 30 Tyr Trp Ser Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Tyr Val Phe His Ser Gly Ile Thr Asn Tyr Ser Pro Ser Leu Lys 50 55 60 Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Lys Ser Val Thr Ala Val Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Lys Lys Ser Gly Thr Ser Lys Lys Pro Ile Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser Gly Gly Gly Ser Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr 130 135 140 Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Ile Thr Ile 145 150 155 160 Thr Cys Arg Ala Ser Gln Ser Val Gly Thr Tyr Val Asn Trp Tyr Gln 165 170 175 His Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Asp Ala Ser Asn 180 185 190 Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Val Thr 195 200 205 Asp Phe Thr Leu Thr Ile Thr Asn Leu Gln Pro Glu Asp Ser Ala Thr 210 215 220 Tyr Tyr Cys Leu Gln His Lys Asn Tyr Pro Leu Thr Phe Gly Gly Gly 225 230 235 240 Thr Lys Val Glu Ile Lys Arg 245 <210> 29 <211> 251 <212> PRT <213> Artificial Sequence <220> <223> B1 < 400> 29 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Phe Asn Phe Asp Glu Tyr 20 25 30 Ala Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Phe Ile Arg Ser Lys Ala Tyr Arg Glu Thr Thr Glu Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Cys Thr Ile Ser Arg Asp Asp Ser Lys Ser Ile 65 70 75 80 Ala Tyr Leu Leu Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala Arg Gly His Thr Pro Gln Arg Pro Tyr Phe Asp Glu Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Ser Ser Gly 115 120 125 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile 130 135 140 Gln Met Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly Asp Arg 145 150 155 160 Ile Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Gly Thr Tyr Val Asn 165 170 175 Trp Tyr Gln Gln Lys Leu Gly Lys Ala Pro Lys Leu Leu Ile Tyr Thr 180 185 190 Ala Ser Thr Leu Gln Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly 195 200 205 Ser Gly Thr Glu Phe Thr Leu Thr Ile Asn Gly Leu Gln Pro Glu Asp 210 215 220 Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Arg Pro His Phe Leu Thr 225 230 235 240 Phe Gly Gly Gly Thr Gln Leu Thr Val Leu Gly 245 250 <210> 30 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> CL <400> 30 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 1 5 10 15 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 65 70 75 80 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 31 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> CH1 <400> 31 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val <210> 32 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> CH2 <400> 32 Glu Pro Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 20 25 30 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 35 40 45 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 50 55 60 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 65 70 75 80 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 85 90 95 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 100 105 110 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 115 120 125 <210> 33 <211> 107 <212> PRT < 213> Artificial Sequence <220> <223> CH3 <400> 33 Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95 Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105 <210> 34 <211> 41 <212> PRT <213> homo sapiens <400> 34 Met Gly Phe Asn Leu Thr Leu Ala Lys Leu Pro Asn Asn Glu Leu His 1 5 10 15 Gly Gln Glu Ser His Asn Ser Gly Asn Arg Ser Asp Gly Pro Gly Lys 20 25 30 Asn Thr Thr Leu His Asn Glu Phe Asp 35 40 <210> 35 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> GPR87(N)-GST <400> 35 Met Gly Phe Asn Leu Thr Leu Ala Lys Leu Pro Asn Asn Glu Leu His 1 5 10 15 Gly Gln Glu Ser His Asn Ser Gly Asn Arg Ser Asp Gly Pro Gly Lys 20 25 30 Asn Thr Thr Leu His Asn Glu Phe Asp Met Ser Pro Ile Leu Gly Tyr 35 40 45 Trp Lys Ile Lys Gly Leu Val Gln Pro Thr Arg Leu Leu Leu Glu Tyr 50 55 60 Leu Glu Glu Lys Tyr Glu Glu His Leu Tyr Glu Arg Asp Glu Gly Asp 65 70 75 80 Lys Trp Arg Asn Lys Lys Phe Glu Leu Gly Leu Glu Phe Pro Asn Leu 85 90 95 Pro Tyr Tyr Ile Asp Gly Asp Val Lys Leu Thr Gln Ser Met Ala Ile 100 105 110 Ile Arg Tyr Ile Ala Asp Lys His Asn Met Leu Gly Gly Cys Pro Lys 115 120 125 Glu Arg Ala Glu Ile Ser Met Leu Glu Gly Ala Val Leu Asp Ile Arg 130 135 140 Tyr Gly Val Ser Arg Ile Ala Tyr Ser Lys Asp Phe Glu Thr Leu Lys 145 150 155 160 Val Asp Phe Leu Ser Lys Leu Pro Glu Met Leu Lys Met Phe Glu Asp 165 170 175 Arg Leu Cys His Lys Thr Tyr Leu Asn Gly Asp His Val Thr His Pro 180 185 190 Asp Phe Met Leu Tyr Asp Ala Leu Asp Val Val Leu Tyr Met Asp Pro 195 200 205 Met Cys Leu Asp Ala Phe Pro Lys Leu Val Cys Phe Lys Lys Arg Ile 210 215 220 Glu Ala Ile Pro Gln Ile Asp Lys Tyr Leu Lys Ser Ser Lys Tyr Ile 225 230 235 240 Ala Trp Pro Leu Gln Gly Trp Gln Ala Thr Phe Gly Gly Gly Asp His 245 250 255 Pro Pro Lys Arg Ser 260 <210> 36 <211> 208 <212> PRT <213> Artificial Sequence <220 > <223> GPR87(N)-Streptavidin <400> 36 Met Gly Phe Asn Leu Thr Leu Ala Lys Leu Pro Asn Asn Glu Leu His 1 5 10 15 Gly Gln Glu Ser His Asn Ser Gly Asn Arg Ser Asp Gly Pro Gly Lys 20 25 30 Asn Thr Thr Leu His Asn Glu Phe Asp Asp Pro Ser Lys Glu Ser Lys 35 40 45 Ala Gln Ala Ala Val Ala Glu Ala Gly Ile Thr Gly Thr Trp Tyr Asn 50 55 60 Gln Leu Gly Ser Thr Phe Ile Val Thr Ala Asn Pro Asp Gly Ser Leu 65 70 75 80 Thr Gly Thr Tyr Glu Ser Ala Val Gly Asn Ala Glu Ser Arg Tyr Val 85 90 95 Leu Thr Gly Arg Tyr Asp Ser Thr Pro Ala Thr Asp Gly Ser Gly Thr 100 105 110 Ala Leu Gly Trp Thr Val Ala Trp Lys Asn Asn Tyr Arg Asn Ala His 115 120 125 Ser Ala Thr Thr Trp Ser Gly Gln Tyr Val Gly Gly Ala Glu Ala Arg 130 135 140 Ile Asn Thr Gln Trp Leu Leu Thr Ser Gly Thr Thr Ala Ala Asn Ala 145 150 155 160 Trp Lys Ser Thr Leu Val Gly His Asp Thr Phe Thr Lys Val Lys Pro 165 170 175 Ser Ala Ala Ser Ile Asp Ala Ala Lys Lys Ala Gly Val Asn Asn Gly 180 185 190Asn Pro Leu Asp Ala Val Gln Gln Gly Ser His His His His His His 195 200 205

Claims (20)

An antibody specific to the extracellular domain of GPR87 (G-protein coupled receptor 87) or a fragment thereof with immunological activity, (i) CDRH1 of SEQ ID NO: 1, CDRH2 of SEQ ID NO: 3, and SEQ ID NO: 5 VH domain containing CDRH3; and an antibody comprising a V domain comprising CDRL1 of SEQ ID NO: 7, CDRL2 of SEQ ID NO: 8, and CDRL3 of SEQ ID NO: 10, and a VL domain comprising CDRL3 of SEQ ID NO: 10, or an immunologically active fragment thereof; or (ii) a VH domain comprising CDRH1 of SEQ ID NO: 2, CDRH2 of SEQ ID NO: 4, and CDRH3 of SEQ ID NO: 6; and a V domain comprising CDRL1 of SEQ ID NO: 7, CDRL2 of SEQ ID NO: 9, and CDRL3 of SEQ ID NO: 11. The antibody or fragment having immunological activity according to claim 1, which specifically binds to an epitope containing the amino acid sequence of SEQ ID NO: 34. delete delete The method of claim 1, wherein FR1 comprising the amino acid sequence of SEQ ID NO: 12 or 13, FR2 comprising the amino acid sequence of SEQ ID NO: 14 or 15, FR3 comprising the amino acid sequence of SEQ ID NO: 16 or 17, and SEQ ID NO: 18 or An antibody specific for the extracellular region of GPR87, or a fragment thereof with immunological activity, containing a VH domain including FR4 containing the amino acid sequence of 19. The method of claim 1, wherein FR1 comprising the amino acid sequence of SEQ ID NO: 20 or 21, FR2 comprising the amino acid sequence of SEQ ID NO: 22 or 23, FR3 comprising the amino acid sequence of SEQ ID NO: 24 or 25, and SEQ ID NO: 26 or An antibody specific for the extracellular region of GPR87, or a fragment thereof with immunological activity, containing a VL domain containing FR4 containing the amino acid sequence of 27. delete The method of claim 1, wherein i) FR1 comprising the amino acid sequence of SEQ ID NO: 12, CDRH1 comprising the amino acid sequence of SEQ ID NO: 1, FR2 comprising the amino acid sequence of SEQ ID NO: 14, and comprising the amino acid sequence of SEQ ID NO: 3 A VH domain comprising CDRH2, FR3 comprising the amino acid sequence of SEQ ID NO: 16, CDRH3 comprising the amino acid sequence of SEQ ID NO: 5, and FR4 comprising the amino acid sequence of SEQ ID NO: 18; and
ii) FR1 containing the amino acid sequence of SEQ ID NO: 20, CDRL1 containing the amino acid sequence of SEQ ID NO: 7, FR2 containing the amino acid sequence of SEQ ID NO: 22, CDRL2 containing the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 24 Specific to the extracellular region of GPR87, comprising a V domain containing FR3 containing the amino acid sequence, CDRL3 containing the amino acid sequence of SEQ ID NO: 10, and a VL domain containing FR4 containing the amino acid sequence of SEQ ID NO: 26 Antibodies or immunologically active fragments thereof; or
i) FR1 containing the amino acid sequence of SEQ ID NO: 13, CDRH1 containing the amino acid sequence of SEQ ID NO: 2, FR2 containing the amino acid sequence of SEQ ID NO: 15, CDRH2 containing the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 17 A VH domain comprising FR3 comprising the amino acid sequence, CDRH3 comprising the amino acid sequence of SEQ ID NO: 6, and FR4 comprising the amino acid sequence of SEQ ID NO: 19; and
ii) FR1 containing the amino acid sequence of SEQ ID NO: 21, CDRL1 containing the amino acid sequence of SEQ ID NO: 7, FR2 containing the amino acid sequence of SEQ ID NO: 23, CDRL2 containing the amino acid sequence of SEQ ID NO: 9, SEQ ID NO: 25 Specific to the extracellular region of GPR87, comprising a V domain containing FR3 containing the amino acid sequence, CDRL3 containing the amino acid sequence of SEQ ID NO: 11, and a VL domain containing FR4 containing the amino acid sequence of SEQ ID NO: 27 Antibodies or fragments thereof with immunological activity. The antibody or fragment having immunological activity according to claim 1, which is specific for the extracellular region of GPR87 and contains the amino acid sequence of SEQ ID NO: 28. The antibody or fragment having immunological activity according to claim 1, which is specific for the extracellular region of GPR87 and contains the amino acid sequence of SEQ ID NO: 29. The method of claim 1, wherein the fragment with immunological activity is Fab, Fd, Fab', dAb, F(ab'), F(ab') ₂ , scFv (single chain fragment variable), Fv, single chain antibody, Fv An antibody specific for the extracellular region of GPR87, or a fragment thereof with immunological activity, selected from the group consisting of dimers, complementarity-determining region fragments, humanized antibodies, chimeric antibodies, and diabodies. An isolated nucleic acid molecule encoding the antibody of claim 1 or an immunologically active fragment thereof. A vector comprising the isolated nucleic acid molecule of claim 12. A host cell transformed with the vector of claim 13. A pharmaceutical composition for the treatment or prevention of cancer comprising the antibody of claim 1 or an immunologically active fragment thereof as an active ingredient. The method of claim 15, wherein the cancer is brain tumor, melanoma, myeloma, non-small cell lung cancer, oral cancer, liver cancer, stomach cancer, colon cancer, breast cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cervical cancer, ovarian cancer, and colon cancer. , small intestine cancer, rectal cancer, fallopian tube carcinoma, anal cancer, endometrial carcinoma, vaginal carcinoma, vulvar carcinoma, Hodgkin's disease, esophageal cancer, lymph node cancer, bladder cancer, gallbladder cancer, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer. , soft tissue sarcoma, urethral cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocytic lymphoma, renal or ureteral cancer, renal cell carcinoma, renal pelvic carcinoma, central nervous system tumor, primary central nervous system lymphoma, spinal cord tumor, brainstem glioma. and a pharmaceutical composition for treating or preventing cancer, which is selected from the group consisting of pituitary adenoma. The pharmaceutical composition for treating or preventing cancer according to claim 15, which inhibits cancer invasion or metastasis. A composition for cancer diagnosis comprising an antibody specific for the extracellular region of GPR87 of claim 1 or a fragment thereof with immunological activity. delete a) cultivating a host cell transformed with a vector containing an isolated nucleic acid molecule encoding an antibody specific for the extracellular region of GPR87 of claim 1 or a fragment having immunological activity thereof; and
b) A method of producing an antibody or an immunologically active fragment thereof specific to the extracellular region of GPR87, comprising the step of recovering the antibody or an immunologically active fragment thereof from a host cell culture.