Anti-CXCR2 antibody and use thereof

Abstract

The present invention relates to an anti-CXCR2 antibody, and a use thereof. The anti-CXCR2 antibody or an antigen-binding fragment thereof of the present invention can specifically bind to cancer cells expressing CXCR2, and exhibits an anti-tumor effect by inhibiting the CXCL8 / CXCR2 signaling pathway, which plays an important role in the formation and growth of tumors, and thus can be effectively used for the treatment of CXCR2-overexpressing cancer patients. In addition, the anti-CXCR2 antibody or an antigen-binding fragment thereof of the present invention specifically binds to CXCR2, thereby enabling the diagnosis of CXCR2-overexpressing cancer.

Description

Anti-CXCR2 antibodies and uses thereof

[0001] The present invention relates to anti-CXCR2 antibodies and uses thereof.

[0002] Chemokines, or chemoattractant cytokines, typically comprise a family of small-molecular-weight (~8 to 10 KDa) secreted inducible molecules that function as activating factors and chemoattractants for leukocytes and can regulate angiogenesis, wound healing, and tumorigenesis. Due to their role in immune modulation and inflammation regulation, most information on chemokine function comes from studies of the immune system. Their roles in many other biological systems are gradually being elucidated.

[0003] Chemokines are typically classified into four subfamilies based on the number of conserved cysteine ​​residues at their amino terminus. Most chemokines fall into two major subfamilies with four cysteine ​​residues. These subfamilies are typically classified based on the presence or absence of an amino acid sequence between the two amino-terminal cysteine ​​residues, hence the designations CC and CXC chemokines. CXC chemokines are typically restricted to higher vertebrates and are further classified based on the presence or absence of a glutamate-lysine-arginine (ELR) motif at the amino terminus, typically adjacent to the first cysteine ​​residue. CXCL1, previously known as Gro-α, is a member of the ELR family of CXC chemokines, whose preferred receptor is CXCR2.

[0004] Each chemokine receptor typically binds to a single class of chemokines, but can bind with high affinity to several members of the same class. Furthermore, a single chemokine can bind to multiple different chemokine receptors. For example, CXCR2 can bind to CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and CXCL8.

[0005] The above CXCR2 is a type A GPCR belonging to the chemokine receptor family with a size of ~41 kDa. CXCR2 is the only high-affinity receptor for all proangiogenic chemokines (CXCL1-3, CXCL5-8). CXCL6 and CXCL8 (IL-8) also interact with CXCR1 to induce chemotactic effects. Physiologically, CXCR2 is involved in the mobilization and recruitment of leukocytes (particularly neutrophils) from the bone marrow to sites of inflammation and in the migration of endothelial cells in angiogenesis.

[0006] Furthermore, CXCR2 has been reported to be expressed in various cell types associated with inflammation and cancer, including neutrophils, granulocytes, endothelial cells, primary cancer cells, and cancer stem cells. Furthermore, the CXCLs / CXCR2 signaling axis has been reported to be a signaling pathway that plays a crucial role in various cancers. CXCR2 is highly expressed in several cancer types and is also a poor biomarker for cancer. Furthermore, numerous studies have reported that CXCR2 controls cancer cell proliferation through downstream signal activation or signal exchange with other cell surface receptors. Furthermore, CXCR2-expressing cancer cells migrate along the CXCL8 concentration gradient. Furthermore, in vitro and in vivo studies have reported that CXCL8 / CXCR2 signaling is involved in apoptosis and angiogenesis. Therefore, inhibition or control of CXCR2 signaling is crucial for achieving antitumor effects.

[0007] In this regard, the present inventors have made extensive efforts to develop an anti-CXCR2 antibody, and have newly produced a human antibody capable of specifically binding to the CXCR2 protein and inhibiting the CXCL8 signal pathway from an antibody library, and have confirmed that the antibody effectively inhibits tumor formation in CXCR2-overexpressing cancer cells, thereby completing the present invention.

[0008] One object of the present invention is to provide an anti-CXCR2 antibody or an antigen-binding fragment thereof.

[0009] Another object of the present invention is to provide a polynucleotide encoding the anti-CXCR2 antibody or an antigen-binding fragment thereof, an expression vector comprising the polynucleotide, and a transformant into which the expression vector has been introduced.

[0010] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising the anti-CXCR2 antibody or an antigen-binding fragment thereof, a polynucleotide encoding the anti-CXCR2 antibody or an antigen-binding fragment thereof, or an expression vector comprising the polynucleotide.

[0011] Another object of the present invention is to provide an anticancer adjuvant pharmaceutical composition comprising, as an active ingredient, the anti-CXCR2 antibody or an antigen-binding fragment thereof, a polynucleotide encoding the anti-CXCR2 antibody or an antigen-binding fragment thereof, or an expression vector comprising the polynucleotide.

[0012] Another object of the present invention is to provide a method for treating cancer, comprising the step of administering to a subject the anti-CXCR2 antibody or an antigen-binding fragment thereof, a polynucleotide encoding the anti-CXCR2 antibody or an antigen-binding fragment thereof, or an expression vector comprising the polynucleotide.

[0013] Another object of the present invention is to provide a method for inhibiting the proliferation of tumor cells expressing CXCR2 protein, comprising the step of contacting the tumor cells expressing CXCR2 protein with the anti-CXCR2 antibody or an antigen-binding fragment thereof.

[0014] Another object of the present invention is to provide a composition for diagnosing CXCR2 overexpressing cancer, comprising the anti-CXCR2 antibody or an antigen-binding fragment thereof, a polynucleotide encoding the anti-CXCR2 antibody or an antigen-binding fragment thereof, or an expression vector comprising the polynucleotide.

[0015] Another object of the present invention is to provide a diagnostic kit for CXCR2 overexpressing cancer comprising the composition and instructions.

[0016] Another object of the present invention is to provide a method for providing information necessary for diagnosing CXCR2 overexpressing cancer, comprising the steps of: (a) contacting a biological sample isolated from an individual suspected of having CXCR2 overexpressing cancer with the anti-CXCR2 antibody or antigen-binding fragment thereof, a polynucleotide encoding the anti-CXCR2 antibody or antigen-binding fragment thereof, or an expression vector comprising the polynucleotide; (b) measuring the expression level of CXCR2 protein from the biological sample; and (c) determining that the individual has CXCR2 overexpressing cancer if the expression level of the CXCR2 protein measured in step (b) is higher than that of a control group.

[0017] To achieve the above-mentioned purpose, the present invention relates to an anti-CXCR2 antibody or an antigen-binding fragment thereof, comprising any one of the heavy chain variable region and the light chain variable region selected from the following 1) and 2):

[0018] 1) a heavy chain variable region comprising a CDR1 region consisting of an amino acid sequence of SEQ ID NO: 7, a CDR2 region consisting of an amino acid sequence of SEQ ID NO: 8, and a CDR3 region consisting of an amino acid sequence of SEQ ID NO: 9, and a light chain variable region comprising a CDR1 region consisting of an amino acid sequence of SEQ ID NO: 10, a CDR2 region consisting of an amino acid sequence of SEQ ID NO: 11, and a CDR3 region consisting of an amino acid sequence of SEQ ID NO: 12; and

[0019] 2) A heavy chain variable region comprising a CDR1 region consisting of the amino acid sequence of SEQ ID NO: 19, a CDR2 region consisting of the amino acid sequence of SEQ ID NO: 20, and a CDR3 region consisting of the amino acid sequence of SEQ ID NO: 21, and a light chain variable region comprising a CDR1 region consisting of the amino acid sequence of SEQ ID NO: 22, a CDR2 region consisting of the amino acid sequence of SEQ ID NO: 23, and a CDR3 region consisting of the amino acid sequence of SEQ ID NO: 24.

[0020] In one embodiment of the present invention, the anti-CXCR2 antibody or antigen-binding fragment thereof may include 1) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 25 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 26; or 2) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 27 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 28.

[0021] In one embodiment of the present invention, the anti-CXCR2 antibody or antigen-binding fragment thereof may have an activity of inhibiting the growth or metastasis of cancer cells expressing CXCR2 protein and inducing apoptosis.

[0022] In one embodiment of the present invention, the anti-CXCR2 antibody or antigen-binding fragment thereof can specifically bind to CXCR2 (CXC motif chemokine receptor 2).

[0023] In one embodiment of the present invention, the anti-CXCR2 antibody or antigen-binding fragment thereof may have an activity of blocking the binding of CXCL8 (CXC motif chemokine ligand 8) and CXCR2.

[0024] In one embodiment of the present invention, the anti-CXCR2 antibody may be a monoclonal antibody.

[0025] In one embodiment of the present invention, the antigen-binding fragment may be any one selected from Fab, Fab', F(ab')2, scFv, Fv, dsFv, diabody, Fd and Fd'.

[0026] In addition, the present invention for achieving the other purposes mentioned above relates to a polynucleotide encoding the anti-CXCR2 antibody or an antigen-binding fragment thereof.

[0027] In one embodiment of the present invention, the polynucleotide may be composed of any one sequence selected from SEQ ID NO: 30 and SEQ ID NO: 32.

[0028] In addition, the present invention for achieving another purpose mentioned above relates to an expression vector comprising the polynucleotide.

[0029] In addition, the present invention for achieving another purpose described above relates to a transformant transformed with the expression vector.

[0030] In addition, the present invention for achieving another purpose described above relates to a pharmaceutical composition for preventing or treating cancer, which comprises as an active ingredient the anti-CXCR2 antibody or an antigen-binding fragment thereof, a polynucleotide encoding the anti-CXCR2 antibody or an antigen-binding fragment thereof, or an expression vector comprising the polynucleotide.

[0031] In one embodiment of the present invention, the cancer may be at least one selected from esophageal cancer, stomach cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, rectal cancer, melanoma, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, soft tissue sarcoma, lymphoma, and multiple myeloma blood cancer.

[0032] In addition, the present invention for achieving another purpose described above relates to an anticancer auxiliary pharmaceutical composition comprising, as an active ingredient, the anti-CXCR2 antibody or an antigen-binding fragment thereof, a polynucleotide encoding the anti-CXCR2 antibody or an antigen-binding fragment thereof, or an expression vector comprising the polynucleotide.

[0033] In addition, the present invention for achieving another purpose described above relates to a method for treating cancer, comprising the step of administering to a subject the anti-CXCR2 antibody or an antigen-binding fragment thereof, a polynucleotide encoding the anti-CXCR2 antibody or an antigen-binding fragment thereof, or an expression vector comprising the polynucleotide.

[0034] In addition, the present invention for achieving another purpose described above relates to a method for inhibiting the proliferation of tumor cells expressing CXCR2 protein, comprising the step of contacting tumor cells expressing CXCR2 protein with the anti-CXCR2 antibody or an antigen-binding fragment thereof.

[0035] In addition, the present invention for achieving another purpose described above relates to a composition for diagnosing CXCR2 overexpressing cancer, comprising the anti-CXCR2 antibody or an antigen-binding fragment thereof, a polynucleotide encoding the anti-CXCR2 antibody or an antigen-binding fragment thereof, or an expression vector comprising the polynucleotide.

[0036] In addition, the present invention for achieving the above-mentioned another object relates to a diagnostic kit for CXCR2 overexpressing cancer comprising the composition and instructions.

[0037] In addition, the present invention for achieving the above-mentioned another object relates to a method for providing information necessary for diagnosing CXCR2 overexpressing cancer, comprising the steps of (a) contacting a biological sample isolated from an individual suspected of having CXCR2 overexpressing cancer with the anti-CXCR2 antibody or an antigen-binding fragment thereof, a polynucleotide encoding the anti-CXCR2 antibody or an antigen-binding fragment thereof, or an expression vector comprising the polynucleotide; (b) measuring the expression level of CXCR2 protein from the biological sample; and (c) determining that the subject has CXCR2 overexpressing cancer if the expression level of the CXCR2 protein measured in the step (b) is higher than that of a control group.

[0038] The anti-CXCR2 antibody or antigen-binding fragment thereof of the present invention can specifically bind to cancer cells expressing CXCR2 and exhibit an anti-tumor effect by inhibiting the CXCL8 / CXCR2 signaling pathway, which plays an important role in tumor formation and growth, and thus can be usefully used for the treatment of cancer patients overexpressing CXCR2. In addition, the anti-CXCR2 antibody or antigen-binding fragment thereof of the present invention can enable the diagnosis of CXCR2-overexpressing cancer by specifically binding to CXCR2.

[0039] Figure 1a shows the result of purifying CXCR2(NTD)-hFc recombinant protein, and Figure 1b shows the result of purifying SUMO-6XHis-CXCR2(NTD) recombinant protein.

[0040] Figure 2 shows the results of producing and purifying full-length IgG antibody candidates (#1, #18, #54, and #56) by subcloning individual DNA sequences of variable heavy (VH) and variable light (VL) chains into an IgG expression vector after selecting four unique scFvs (#1, #18, #54, and #56) that bind to hCXCR2 (NTD) through a biopanning procedure.

[0041] Figure 3 is a graph showing that the binding strength of full-length IgG antibody candidates (#18 and #56) and hCXCR2 (NTD) increases in a dose-dependent manner.

[0042] Figure 4 is a graph showing that the binding strength of full-length IgG antibody candidates (#1, #18, #54, and #56) and hCXCR2 (NTD) increases in a dose-dependent manner.

[0043] Figure 5 is a graph showing the individual KD values ​​of hCXCR2 (NTD) and IgG_18 or IgG_56.

[0044] Figure 6 is a FACS analysis result showing that IgG_18 or IgG_56 according to one embodiment of the present invention strongly binds to HEK293 cells expressing hCXCR2.

[0045] Figure 7 is a result showing the binding affinity of IgG_18 or IgG_56 to HEK293 cells expressing hCXCR1-GFP, hCXCR2-GFP, rhCXCR2-GFP, or mCXCR2-GFP according to one embodiment of the present invention.

[0046] Figure 8 shows the results of measuring the intracellular calcium level induced by CXCL8 after treating HEK293 cells expressing CXCR2 with IgG_18 or IgG_56 according to one embodiment of the present invention together with CXCL8.

[0047] Figure 9 is a Western blot result showing changes in the expression levels of p-ERK and p-AKT when CXCL8 was treated in HEK293 cells expressing CXCR2 in the presence of IgG_18 or IgG_56 according to one embodiment of the present invention.

[0048] Figure 10 is a result showing that IgG_18 or IgG_56 according to one embodiment of the present invention strongly binds to various cancer cell lines such as pancreatic cancer (PANC-1, BxPC-3, AsPC-1), ovarian cancer (Hey A8, SKOV-3, OVCAR-3), colon cancer (SNU-C2A), and lung cancer (A427).

[0049] Figure 11 is a result showing that IgG_18 or IgG_56 according to one embodiment of the present invention inhibits the increase in AsPC-1 cell proliferation by CXCL8.

[0050] Figure 12 is a result showing that IgG_18 or IgG_56 according to one embodiment of the present invention increases the number of AsPC-1 cells passing through the membrane by CXCL8, i.e. reduces the migration of AsPC-1 cells. *** indicates a significant difference at p<0.001 (Student's t test) compared to the control group (Mock).

[0051] Figure 13 is a result showing that IgG_18 or IgG_56 according to one embodiment of the present invention increases the death of AsPC-1 cells reduced by CXCL8.

[0052] AC of FIG. 14 is a result showing that the tumor volume, size, and weight of mice injected with IgG_18 or IgG_56 according to one embodiment of the present invention are significantly reduced compared to the control group (mice injected with PBS), and D of FIG. 14 is a result showing that the body weight of mice injected with IgG_18 or IgG_56 according to one embodiment of the present invention is not significantly different from the control group. ** and *** indicate that there is a significant difference at p<0.01 and p<0.001, respectively, compared to the control group (Dunnett's test).

[0053] Hereinafter, the present invention will be described in detail.

[0054] The present invention provides an anti-CXCR2 antibody or an antigen-binding fragment thereof that specifically binds to CXCR2 (CXC motif chemokine receptor 2). The above CXCR2 is characterized in that it is overexpressed in CXCR2-overexpressing cancer, preferably at least one cancer selected from esophageal cancer, gastric cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, rectal cancer, melanoma, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, soft tissue sarcoma, lymphoma, and multiple myeloma blood cancer, more preferably at least one cancer selected from pancreatic cancer, ovarian cancer, colon cancer, colon cancer, rectal cancer, lung cancer, melanoma, and prostate cancer, more preferably at least one cancer selected from pancreatic cancer, ovarian cancer, colon cancer, and lung cancer.

[0055] The above CXCR2 plays a role in increasing cancer cell growth by regulating cancer cell metastasis and angiogenesis. Specifically, suppression of CXCR2 expression and defects in cancer cells not only suppress angiogenesis, but also reduce cancer cell growth and metastasis and induce apoptosis. CXCR2 is highly expressed in pancreatic, ovarian, colon, rectal, lung, melanoma, and prostate cancer, and its expression is known to increase during inflammation.

[0056] The present inventors identified an antibody that specifically recognizes CXCR2 and elucidated the amino acid sequence of its heavy chain variable region, the amino acid sequence of its light chain variable region, and the coding nucleotide sequence.

[0057] In addition, the present invention provides, as a preferred embodiment, an anti-CXCR2 antibody or an antigen-binding fragment thereof that specifically binds to CXCR2, thereby being able to specifically bind to cancer cells expressing CXCR2 and exhibiting an anti-tumor effect by inhibiting the CXCL8 / CXCR2 signal transduction pathway that plays an important role in tumor formation and growth, and thus can be usefully used for the treatment of cancer patients overexpressing CXCR2. In addition, by specifically detecting CXCR2, it can enable accurate diagnosis of CXCR2-overexpressing cancer cells and / or tissues.

[0058] As used herein, the term "antibody" refers to a protein molecule that acts as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that immunologically has reactivity with a specific antigen, and includes polyclonal antibodies, monoclonal antibodies, complete antibody forms, as well as antigen-binding fragments (antibody fragments) of antibody molecules. In addition, the term includes chimeric antibodies, humanized antibodies, human antibodies, and bivalent or multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies, etc.), diabodies, triabodies, and tetrabodies.

[0059] The above "complete antibody (full-length antibody)" has a structure having two full-length light chains and two full-length heavy chains, each light chain being linked to a heavy chain by a disulfide bond. The complete antibody includes IgA, IgD, IgE, IgM, and IgG, and IgG is divided into subtypes, including IgG1, IgG2, IgG3, and IgG4.

[0060] As used herein, the term "antigen-binding fragment of an antibody" refers to a fragment that possesses antigen-antibody binding function within a complete antibody molecule, and includes Fab, Fab', F(ab')2, scFv, Fv, dsFv, diabody, Fd, and Fd'. The Fab has a structure having variable regions of the light and heavy chains, a constant region of the light chain, and the first constant region (CH1 domain) of the heavy chain, and has one antigen-binding site. 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 CH1 domain. F(ab')2 antibody is generated when cysteine ​​residues in the hinge region of Fab' form a disulfide bond. Fv (variable fragment) refers to the smallest antibody fragment that has only the heavy chain variable region and the light chain variable region. A double-chain Fv (dsFv) has a heavy chain variable region and a light chain variable region linked by a disulfide bond, and a single-chain Fv (scFv) has a heavy chain variable region and a light chain variable region covalently linked by a peptide linker or directly linked at the C-terminus, so that it can form a dimer-like structure like a double-chain Fv. A diabody is a complex of two or more polypeptide chains or proteins, each comprising at least one VL and VH domain or fragment thereof, wherein both domains are contained within a single polypeptide chain. In some embodiments, a diabody comprises a molecule comprising an Fc or hinge-Fc domain. The polypeptide chains of such complexes may be identical or different, i.e., the diabody may be a mono- or hetero-multimer.

[0061] These antigen-binding fragments can be obtained using proteolytic enzymes (for example, a complete antibody can be restricted to papain to obtain a Fab fragment, and a pepsin cleavage can obtain an F(ab')2 fragment), or preferably, can be produced using genetic recombination techniques.

[0062] As used herein, the term “heavy chain” refers to a full-length heavy chain and fragments thereof, which comprise a variable domain VH and three constant domains CH1, CH2 and CH3, which comprise an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. In addition, the term “light chain” as used herein refers to a full-length light chain and fragments thereof, which comprise a variable domain VL and a constant domain CL, which comprise an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen.

[0063] As used herein, the term "monoclonal antibody" refers to an antibody molecule of a single molecular composition obtained from a substantially identical antibody population, and such monoclonal antibody exhibits a single binding specificity and affinity for a specific epitope. Typically, immunoglobulins have heavy and light chains, each of which comprises a constant region and a variable region (also known as a domain). The variable regions of the light and heavy chains comprise three variable regions called complementarity-determining regions (hereinafter referred to as "CDRs") and four framework regions (FRs). The CDRs are primarily responsible for binding to epitopes of an antigen. The CDRs of each chain are typically named CDR1, CDR2, and CDR3, starting from the N-terminus, and are also identified by the chain on which the particular CDR is located.

[0064] As used herein, the term “complementarity determining region (CDR)” refers to the amino acid sequence of the hypervariable region of the immunoglobulin heavy and light chains (Kabat et al. Sequences of Proteins of Immunological Interest, 4th Ed., US Department of Health and Human Services, National Institutes of Health (1987)). The heavy chains (CDRH1, CDRH2, and CDRH3) and light chains (CDRL1, CDRL2, and CDRL3) each contain three CDRs, which provide key contact residues for antibody binding to an antigen or epitope.

[0065] Meanwhile, the monoclonal antibody may be a chimeric antibody or humanized antibody with reduced immunogenicity for application to the human body as described above, or may be a human antibody.

[0066] In the present invention, the term "chimeric antibody" refers to an antibody in the form of a recombinant DNA technology that recombines the variable region of an antibody of a species other than humans, such as a mouse or chicken, with the constant region of a human antibody. Since the immune response of the chimeric antibody is greatly improved compared to antibodies of a species other than humans, such as a mouse or chicken, it can be used clinically.

[0067] In the present invention, the term "humanized antibody" means an antibody in the form of a human antibody in which all or part of the CDR sequence of a monoclonal antibody that is foreign to humans, such as a mouse or chicken, is grafted onto the human antibody. For example, a humanized variable region may be produced by recombining the CDRs of a chicken or mouse monoclonal antibody with a FR derived from a human antibody, and then recombining this with a constant region of a desired human antibody, but is not limited thereto.

[0068] As used herein, the term "human antibody" refers to an antibody produced by a human or human cell, or an antibody derived from a non-human source utilizing human antibody repertoires or other human antibody coding sequences, which has an amino acid sequence corresponding to the amino acid sequence of that antibody.

[0069] As used herein, the term "anti-CXCR2 antibody" refers to an antibody capable of specifically binding to CXCR2. The antibody that specifically binds to the CXCR2 protein includes, without limitation, any antibody that binds to CXCR2 and inhibits the biological activity of CXCR2. In addition, the form of the antibody may include, as described above, a complete antibody, a monoclonal antibody, and an antigen-binding fragment, and may be, but is not limited to, a chimeric antibody, a humanized antibody, or a human antibody. In addition, the antibody of the present invention specifically binds to CXCR2, inhibits signal transduction by CXCR2, and thus inhibits biological activity, so that it is effective in treating cancers involving CXCR2, specifically, cancers overexpressing CXCR2, preferably at least one cancer selected from esophageal cancer, gastric cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, rectal cancer, melanoma, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, soft tissue sarcoma, lymphoma, and multiple myeloma blood cancers, more preferably at least one cancer selected from pancreatic cancer, ovarian cancer, colon cancer, colon cancer, rectal cancer, lung cancer, melanoma, and prostate cancer, more preferably pancreatic cancer, ovarian cancer, and colon cancer. It can be usefully used in the prevention or treatment of one or more types of cancer selected from lung cancer.In addition, since overexpression of CXCR2 has been reported as a specific phenomenon in CXCR2-overexpressing cancer, the antibody of the present invention capable of specifically binding to CXCR2 is used to treat CXCR2-overexpressing cancer, preferably one or more cancers selected from esophageal cancer, gastric cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, rectal cancer, melanoma, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, soft tissue sarcoma, lymphoma, and multiple myeloma blood cancer, more preferably one or more cancers selected from pancreatic cancer, ovarian cancer, colon cancer, colon cancer, rectal cancer, lung cancer, melanoma, and prostate cancer, more preferably pancreatic cancer, ovarian cancer, colon cancer, and Since it has a diagnostic ability with high sensitivity and specificity in the diagnosis of one or more types of cancer selected from lung cancer, it can be usefully used in the diagnosis of the above cancer. In one embodiment of the present invention, an antibody or antigen-binding fragment thereof that specifically binds to CXCR2 of the present invention was produced using CXCR2 as an antigen protein.

[0070] The antibody or antigen-binding fragment thereof that specifically binds to the above CXCR2 may include any one of the heavy chain variable region and light chain variable region selected from 1) and 2) below, but is not limited thereto:

[0071] 1) a heavy chain variable region comprising a CDR1 region consisting of an amino acid sequence of SEQ ID NO: 7, a CDR2 region consisting of an amino acid sequence of SEQ ID NO: 8, and a CDR3 region consisting of an amino acid sequence of SEQ ID NO: 9, and a light chain variable region comprising a CDR1 region consisting of an amino acid sequence of SEQ ID NO: 10, a CDR2 region consisting of an amino acid sequence of SEQ ID NO: 11, and a CDR3 region consisting of an amino acid sequence of SEQ ID NO: 12; and

[0072] 2) A heavy chain variable region comprising a CDR1 region consisting of the amino acid sequence of SEQ ID NO: 19, a CDR2 region consisting of the amino acid sequence of SEQ ID NO: 20, and a CDR3 region consisting of the amino acid sequence of SEQ ID NO: 21, and a light chain variable region comprising a CDR1 region consisting of the amino acid sequence of SEQ ID NO: 22, a CDR2 region consisting of the amino acid sequence of SEQ ID NO: 23, and a CDR3 region consisting of the amino acid sequence of SEQ ID NO: 24.

[0073] In one embodiment of the present invention, the anti-CXCR2 antibody or antigen-binding fragment thereof may include 1) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 25 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 26; or 2) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 27 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 28.

[0074] The anti-CXCR2 antibody or antigen-binding fragment thereof of the present invention includes all mutants that achieve the intended effect of the present invention through one or more mutations, such as substitutions, deletions, inversions or translocations, in the antibody limited to the above sequence, and are included in the scope of protection of the present invention.

[0075] In one embodiment of the present invention, the anti-CXCR2 antibody or antigen-binding fragment thereof may have an activity of inducing apoptosis of cancer cells expressing the CXCR2 protein.

[0076] In one embodiment of the present invention, the anti-CXCR2 antibody or antigen-binding fragment thereof can specifically bind to CXCR2 (CXC motif chemokine receptor 2).

[0077] In one embodiment of the present invention, the anti-CXCR2 antibody or antigen-binding fragment thereof may have an activity of blocking the binding of CXCL8 (CXC motif chemokine ligand 8) and CXCR2.

[0078] In one embodiment of the present invention, the antigen-binding fragment is preferably, but not limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFv, diabody, Fd, Fd', a light or heavy chain comprising the CDR region of the present invention, or a variable domain comprising the CDR region of the present invention.

[0079] The anti-CXCR2 antibody or antigen-binding fragment thereof of the present invention exhibits high apoptotic activity and can suppress tumor formation in CXCR2-overexpressing cancer, preferably one or more cancers selected from esophageal cancer, gastric cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, rectal cancer, melanoma, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, soft tissue sarcoma, lymphoma, and multiple myeloma hematological cancer, more preferably one or more cancers selected from pancreatic cancer, ovarian cancer, colon cancer, colon cancer, rectal cancer, lung cancer, melanoma, and prostate cancer, and more preferably one or more cancers selected from pancreatic cancer, ovarian cancer, colon cancer, and lung cancer.

[0080]

[0081] In another aspect, the present invention provides a polynucleotide encoding the anti-CXCR2 antibody or an antigen-binding fragment thereof.

[0082] The above polynucleotide may be composed of any one sequence selected from SEQ ID NO: 29 to SEQ ID NO: 32, and preferably any one sequence selected from SEQ ID NO: 30 and SEQ ID NO: 32, but is not limited thereto.

[0083] In another aspect, the present invention provides an expression vector comprising the polynucleotide, and a transformant into which the vector has been introduced.

[0084] The expression vector comprising a polynucleotide encoding the anti-CXCR2 antibody provided in the present invention is not particularly limited thereto, but may be a vector capable of replicating and / or expressing the polynucleotide in eukaryotic or prokaryotic cells including mammalian cells (e.g., human, monkey, rabbit, rat, hamster, mouse cells, etc.), plant cells, yeast cells, insect cells, or bacterial cells (e.g., Escherichia coli, etc.), and preferably, may be a vector operably linked to an appropriate promoter so that the nucleotide can be expressed in a host cell, and may include at least one selectable marker. For example, the polynucleotide may be introduced into a phage, plasmid, cosmid, mini-chromosome, virus (e.g., retrovirus, lentivirus, adenovirus, adeno-associated virus, etc.) vector.

[0085] The expression vector comprising a polynucleotide encoding the anti-CXCR2 antibody may be an expression vector comprising a polynucleotide encoding a heavy chain or a light chain of the anti-CXCR2 antibody, or an expression vector comprising both a polynucleotide encoding a heavy chain or a light chain.

[0086] The transformant into which the expression vector provided in the present invention has been introduced is not particularly limited thereto, but may be a bacterial cell such as Escherichia coli, Streptomyces, or Salmonella Typhimurium transformed by the introduction of the expression vector; a yeast cell; a fungal cell such as Pichia pastoris; an insect cell such as Drosophila or Spodoptera Sf9 cell; an animal cell such as CHO (Chinese hamster ovary cells), SP2 / 0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, Bow melanoma cells, HT-1080, BHK (baby hamster kidney cells), HEK (human embryonic kidney cells), PERC.6 (human retinal cells), or a plant cell.

[0087] In the present invention, the term "introduction" refers to a method of delivering a vector containing a polynucleotide encoding the anti-CXCR2 antibody to a host cell. Such introduction can be performed by various methods known in the art, such as calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofectamine, and protoplast fusion. Furthermore, transduction refers to delivering a target substance into a cell using a viral particle by means of infection. In addition, a vector can be introduced into a host cell by gene bombardment, etc. In the present invention, introduction can be used interchangeably with transformation.

[0088] It is preferable that the host cell of the present invention is an 'isolated' host cell.

[0089] The above host cell may be a host cell derived from a mammal other than a human.

[0090]

[0091] In another aspect, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising, as an active ingredient, the anti-CXCR2 antibody or an antigen-binding fragment thereof, a polynucleotide encoding the anti-CXCR2 antibody or an antigen-binding fragment thereof, or an expression vector comprising the polynucleotide.

[0092] Since the “anti-CXCR2 antibody or antigen-binding fragment thereof” of the present invention has already been described above, its description is omitted to avoid excessive duplication.

[0093] In the present invention, the cancer may be at least one cancer selected from esophageal cancer, stomach cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, rectal cancer, melanoma, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, soft tissue sarcoma, lymphoma, and multiple myeloma hematological cancer. Preferably, the cancer may be at least one cancer selected from pancreatic cancer, ovarian cancer, colon cancer, colon cancer, rectal cancer, lung cancer, melanoma, and prostate cancer. More preferably, the cancer may be at least one cancer selected from pancreatic cancer, ovarian cancer, colon cancer, and lung cancer. More preferably, the cancer may be pancreatic cancer.

[0094] As used herein, the term "prevention" may refer to any action that inhibits or delays the onset of cancer by administering the composition. Furthermore, the term "treatment" as used herein may refer to any action that improves or beneficially alters the symptoms of cancer by administering the composition.

[0095] The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier.

[0096] In the present invention, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate a living organism and does not inhibit the biological activity and properties of the administered compound. In a composition formulated as a liquid solution, acceptable pharmaceutical carriers are sterile and biocompatible, and include saline solution, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components. If necessary, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, pills, capsules, granules, or tablets.

[0097] The above pharmaceutical composition may be in various oral or parenteral dosage forms. When formulated, it is prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and these solid preparations are prepared by mixing one or more compounds with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to 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 include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0098] The above pharmaceutical composition may have any one dosage form selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid solutions, emulsions, syrups, sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories.

[0099] The composition of the present invention is administered in a pharmaceutically effective amount.

[0100] As used herein, the term "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dosage level can be determined according to factors including the type and severity of the individual, age, sex, type of cancer, activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents. And it can be administered singly or in multiple doses. It is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects by taking all of the above factors into consideration, and it can be easily determined by those skilled in the art.

[0101] In one embodiment of the present invention, it was confirmed that the anti-CXCR2 antibody of the present invention not only specifically binds to CXCR2, but also inhibits the CXCL8 / CXCR2 signal transduction pathway, thereby inhibiting the growth or metastasis of cancer cells expressing CXCR2, inducing apoptosis, and inhibiting the formation and growth of tumors, thereby indicating that a pharmaceutical composition including the antibody of the present invention can be usefully used in the prevention or treatment of CXCR2-overexpressing cancer.

[0102]

[0103] In another aspect, the present invention provides an anticancer adjuvant pharmaceutical composition comprising, as an active ingredient, the anti-CXCR2 antibody or an antigen-binding fragment thereof, a polynucleotide encoding the anti-CXCR2 antibody or an antigen-binding fragment thereof, or an expression vector comprising the polynucleotide.

[0104] Since the “anti-CXCR2 antibody or antigen-binding fragment thereof” and “cancer” of the present invention have already been described above, their description is omitted to avoid excessive duplication.

[0105] In the present invention, the term “anticancer adjuvant” refers to use as an anticancer adjuvant used in combination with an anticancer drug to maximize the effect of the anticancer drug by increasing sensitivity to the anticancer drug.

[0106] In the present invention, the term “enhancing sensitivity to anticancer drugs” means maximizing the antitumor effect by suppressing anticancer drug resistance, which is the acquisition of non-specific resistance by cancer cells regardless of the structure of the anticancer drug.

[0107] Anticancer agents that can be used in combination with the anticancer adjuvant pharmaceutical composition of the present invention include nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, sorafenib, bevacizumab, cisplatin, cetuximab, viscum album, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumomab tyusetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan, gemcitabine, doxifluridine, pemetrexed, tegafur, capecitabine, gimeracin, oteracil, azacitidine, methotrexate, Uracil, cytarabine, fluorouracil, fludagabine, enocitabine, decitabine, mercaptopurine, thioguanine, cladribine, carmofur, raltitrexed, docetaxel, paclitaxel, irinotecan, belotecan, topotecan, vinorelbine, etoposide, vincristine, vinblastine, teniposide, doxorubicin, idarubicin, epirubicin, mitoxantrone, mitomycin, bleromycin, daunorubicin, dactinomycin, pirarubicin, aclarubicin, pepromycin, temozolomide, busulfan, ifosfamide, cyclophosphamide, melphalan, altretmine, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, lomustine or carmustine, but may include: It is not limited.

[0108]

[0109] In another aspect, the present invention provides a method for treating cancer, comprising the step of administering to a subject the anti-CXCR2 antibody or an antigen-binding fragment thereof, a polynucleotide encoding the anti-CXCR2 antibody or an antigen-binding fragment thereof, or an expression vector comprising the polynucleotide.

[0110] Since the “anti-CXCR2 antibody or antigen-binding fragment thereof” and “cancer” of the present invention have already been described above, their description is omitted to avoid excessive duplication.

[0111] The method for treating the cancer may be a method for treating CXCR2-overexpressing cancer, comprising administering to a subject in need thereof a pharmaceutical composition comprising the anti-CXCR2 antibody or antigen-binding fragment thereof of the present invention, a polynucleotide encoding the anti-CXCR2 antibody or antigen-binding fragment thereof, or an expression vector comprising the polynucleotide, and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier is the same as described above.

[0112] The above-mentioned subject includes mammals, birds, reptiles, amphibians, fish, etc., including cows, pigs, sheep, chickens, dogs, humans, etc., and subjects in which CXCR2-overexpressing cancer is treated by administration of the composition of the present invention are included without limitation. The above-mentioned subject may be a mammal other than a human.

[0113] At this time, the composition may be administered singly or multiple times in a pharmaceutically effective amount. At this time, the composition may be administered in the form of a liquid, powder, aerosol, capsule, enteric-coated tablet, or capsule or suppository. Routes of administration include, but are not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration. However, since peptides are digested when administered orally, oral compositions must be formulated to coat the active agent or protect it from degradation in the stomach. Furthermore, the pharmaceutical composition may be administered by any device capable of transporting the active agent to target cells.

[0114] Since the pharmaceutical composition of the present invention includes an antibody that specifically binds to CXCR2 of the present invention, when the pharmaceutical composition including the antibody is administered into a human body, the occurrence, proliferation, or metastasis of CXCR2-overexpressing cancer can be suppressed or the progression can be prevented, thereby treating CXCR2-overexpressing cancer.

[0115]

[0116] In another aspect, the present invention provides a method for inhibiting the proliferation of tumor cells expressing CXCR2 protein, comprising the step of contacting the tumor cells expressing CXCR2 protein with the anti-CXCR2 antibody or an antigen-binding fragment thereof.

[0117] Since the “anti-CXCR2 antibody or antigen-binding fragment thereof” of the present invention has already been described above, its description is omitted to avoid excessive duplication.

[0118] The above method can be performed in-vivo, ex-vivo or in-vitro, but is preferably performed ex-vivo or in-vitro.

[0119]

[0120] In another aspect, the present invention provides a composition for diagnosing CXCR2 overexpressing cancer, comprising the monoclonal antibody or an antigen-binding fragment thereof, a polynucleotide encoding the monoclonal antibody or an antigen-binding fragment thereof, or an expression vector comprising the polynucleotide.

[0121] Since the “anti-CXCR2 antibody or antigen-binding fragment thereof” of the present invention has already been described above, its description is omitted to avoid excessive duplication.

[0122] The term “overexpression” used to express a cancer in which CXCR2 is overexpressed in the present invention means that, when the expression level of CXCR2 is measured through a suitable expression analysis method (e.g., Western blot analysis, Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine, 108-121, CRC press), the expression level of CXCR2 is 1.5 times or more, preferably 1.7 times or more, more preferably 2 times or more, even more preferably 2.3 times or more, compared to the expression level of CXCR2 in normal cells, or 1.5 times or more, preferably 1.7 times or more, more preferably 2 times or more, even more preferably 2.5 times or more, even more preferably 3 times or more, compared to the expression level of CXCR2 in non-malignant cancer cells.

[0123] The term “CXCR2 overexpressing cancer” of the present invention may be at least one cancer selected from esophageal cancer, stomach cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, rectal cancer, melanoma, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin’s disease, soft tissue sarcoma, lymphoma, and multiple myeloma blood cancer, and preferably may be at least one cancer selected from pancreatic cancer, ovarian cancer, colon cancer, colon cancer, rectal cancer, lung cancer, melanoma, and prostate cancer, and more preferably may be at least one cancer selected from pancreatic cancer, ovarian cancer, colon cancer, and lung cancer.

[0124] In the present invention, the term "diagnosis" means confirming the presence, degree (symptoms), and / or characteristics of a pathological condition. As used herein, "diagnosis of CXCR2-overexpressing cancer" means confirming the pathological condition of CXCR2-overexpressing cancer, such as whether CXCR2-overexpressing cancer has occurred, the degree of CXCR2-overexpressing cancer site, the location of CXCR2-overexpressing cancer, and the degree of CXCR2-overexpressing cancer. For a more accurate diagnosis, it is important to accurately and quickly distinguish CXCR2-overexpressing cancer cells or CXCR2-overexpressing cancer cells and / or tissues from normal cells or normal tissues.

[0125] As a specific example of the present invention, by providing an antibody that specifically binds to CXCR2, the present invention can specifically detect CXCR2 at the protein level, thereby enabling accurate diagnosis of CXCR2-overexpressing cancer cells and / or tissues. Furthermore, a diagnostic composition comprising the CXCR2-specific antibody of the present invention can be used to diagnose diseases related to the presence or level of CXCR2 expression or diseases mediated by CXCR2, such as CXCR2-overexpressing cancer.

[0126]

[0127] In another aspect, the present invention provides a diagnostic kit for CXCR2 overexpressing cancer, comprising the diagnostic composition for CXCR2 overexpressing cancer.

[0128] Since the “anti-CXCR2 antibody or antigen-binding fragment thereof” and “CXCR2-overexpressing cancer” of the present invention have already been described above, their description is omitted to avoid excessive duplication.

[0129] The above diagnostic kit for CXCR2 overexpression cancer may further comprise a composition, solution, or device containing one or more other components suitable for the analysis method. In addition, the diagnostic kit for CXCR2 overexpression cancer may further comprise an instruction manual.

[0130] The present specification provides an antibody that specifically binds to CXCR2, which is overexpressed in CXCR2-overexpressing cancer, thereby providing a useful means for measuring the expression and / or expression level of CXCR2, and thereby can be usefully applied to the diagnosis of CXCR2-overexpressing cancer. In addition, the antibody, when used in combination with various markers, can be used to visualize CXCR2-overexpressing cancer, and in particular, can provide more accurate information in confirming the presence of CXCR2-overexpressing cancer, confirming the location of lesions, and observing the morphology of CXCR2-overexpressing cancer, and can reduce the misdiagnosis rate related to CXCR2-overexpressing cancer and contribute to early diagnosis. In addition, when a bioactive substance such as a drug is conjugated to the antibody, the bioactive substance can be specifically delivered to colon cancer, and thus can be used as a composition for targeted delivery of the bioactive substance to CXCR2-overexpressing cancer.

[0131]

[0132] In another aspect, the present invention provides a method for providing information necessary for diagnosing CXCR2 overexpressing cancer, comprising the steps of: (a) contacting a biological sample isolated from an individual suspected of having CXCR2 overexpressing cancer with the anti-CXCR2 antibody or antigen-binding fragment thereof, a polynucleotide encoding the anti-CXCR2 antibody or antigen-binding fragment thereof, or an expression vector comprising the polynucleotide; (b) measuring the expression level of CXCR2 protein from the biological sample; and (c) determining that the individual has CXCR2 overexpressing cancer if the expression level of the CXCR2 protein measured in step (b) is higher than that of a control group.

[0133] The anti-CXCR2 antibody, CXCR2-overexpressing cancer, subject, and CXCR2 are as described above. A method for providing information necessary for diagnosing the CXCR2-overexpressing cancer comprises reacting an antibody specific for CXCR2 of the present invention with a separated biological sample from a subject suspected of having CXCR2-overexpressing cancer and measuring the expression level of the CXCR2 protein through the formation of an antigen-antibody complex, thereby providing information for diagnosing the CXCR2-overexpressing cancer. Since the CXCR2 is overexpressed in CXCR2-overexpressing cancer cells, if the expression level of CXCR2 is higher than that of a control group such as a normal cell or tissue, the subject can be determined to have CXCR2-overexpressing cancer.

[0134] Providing the information necessary for the above diagnosis can be used as an auxiliary tool for clinicians in diagnosing CXCR2-overexpressing cancer.

[0135] The provision of information necessary for the above diagnosis can be performed by non-clinicians, and clinicians can make a diagnosis based on their own experience by synthesizing the information provided and other test results.

[0136] The term "biological sample" as used herein includes tissues, cells, whole blood, serum, plasma, autopsy samples (brain, skin, lymph nodes, spinal cord, etc.), saliva, urine, cell culture supernatants, ruptured eukaryotic cells, and bacterial expression systems, and is preferably "isolated" from outside the body. These biological samples can be reacted with the antibodies of the present invention, with or without manipulation, to determine the presence of CXCR2 protein, the expression level, or whether the cancer is CXCR2-overexpressing.

[0137] The expression level of the above CXCR2 protein can be achieved by measuring the expression level of the CXCR2 protein bound to the anti-CXCR2 antibody or antigen-binding fragment thereof in the above biological sample through the formation of an antigen-antibody complex, but is not limited thereto.

[0138] In the present invention, the term "antigen-antibody complex" means a combination of a CXCR2 protein antigen in a sample and a monoclonal antibody according to the present invention that recognizes the same, and the formation of such an antigen-antibody complex can be detected by any method selected from the group consisting of a colorimetric method, an electrochemical method, a fluorimetric method, a luminometry method, a particle counting method, a visual assessment method, and a scintillation counting method. However, it is not necessarily limited to these and various applications and applications are possible.

[0139] In the present invention, various labels can be used to detect antigen-antibody complexes. Specific examples include, but are not limited to, enzymes, fluorescent substances, ligands, luminescent substances, microparticles, and radioactive isotopes.

[0140] Enzymes used as detection markers include acetylcholinesterase, alkaline phosphatase, β-D-galactosidase, horseradish peroxidase, β-ratamase, etc., and fluorescent substances include fluorescein, Eu 3+ , Eu 3+ Chelates or cryptates, etc. are included, ligands include biotin derivatives, etc., luminescent materials include acridinium esters, isoluminol derivatives, etc., microparticles include colloidal gold, colored latex, etc., and radioactive isotopes include 57 Co, 3 H, 125 I, 125 Includes I-Bolton Hunter reagents, etc.

[0141] Preferably, the antigen-antibody complex can be detected using an enzyme-linked immunosorbent assay (ELISA). ELISA includes various ELISA methods, such as a direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled secondary antibody that recognizes a capture antibody in a complex of antibodies that recognize the antigen attached to the solid support, a direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of antibodies and antigen attached to the solid support, and an indirect sandwich ELISA using a labeled secondary antibody that recognizes an antibody that is reacted with another antibody that recognizes an antigen in a complex of antibodies and antigen attached to the solid support.

[0142] The above monoclonal antibodies may have a detection label, and if they do not have a detection label, these monoclonal antibodies can be captured and identified by treating them with another antibody having a detection label.

[0143] In one embodiment of the present invention, it was confirmed that the anti-CXCR2 antibody of the present invention specifically recognizes CXCR2 using an antigen-antibody reaction, thereby showing that the antibody of the present invention can be usefully used in the diagnosis of CXCR2-overexpressing cancer.

[0144]

[0145] Hereinafter, the anti-CXCR2 antibody or antigen-binding fragment thereof according to the present invention will be described in detail with examples.

[0146]

[0147] <Example>

[0148] Experimental materials and methods

[0149] Cell culture and transformation

[0150] HEK293T and Panc-1 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics, respectively. BxPC-3, AsPC-1, Hey A8, and SNU-C2A cells were cultured in RPMI-1640 medium supplemented with 10% FBS and antibiotics, respectively. HEK293, SKOV-3, and A427 cells were maintained in EMEM, McCoy's 5a, and MEM medium, respectively. OVCAR-3 cells were cultured in RPMI-1640 medium supplemented with 20% FBS. For antigen and antibody production, 293F cells or CHO cells were maintained in FreeStyleTM 293F medium or ExpiCHO expression medium (Thermo Fisher Scientific).

[0151] Geneticin was added to the cell culture medium to establish and maintain stable cell lines. In addition, plasmids were transfected using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen).

[0152]

[0153] DNA vector production

[0154] Most expression vectors were constructed using Gateway cloning technology (Invitrogen). Briefly, human CXCR1, human CXCR2, mouse CXCR2, and rhesus macaque CXCR2 cDNAs were amplified by PCR and subcloned into the pENTR-D / TOPO vector. Each cDNA in the TOPO vector was recombined into the pcDNA expression vector containing a C-terminal 3xFLAG-3xHA or GFP tag (Invitrogen). General procedures were performed according to the manufacturer's instructions and a previous study [Song Y.Bet et al., Trafficking-defective mutant PROKR2 cycles between endoplasmic reticulum and Golgi to attenuate endoplasmic reticulum stress. Proc Natl Acad Sci U S A. 2022 Feb 22;119(8):e2102248119.].

[0155] First, to produce CXCR2(NTD)-hFC protein as an antigen, the DNA sequence encoding the N-terminal domain of human CXCR2 (1-48 aa) was inserted into the pcDNA3.1 mammalian expression vector. Specifically, the pcDNA3.1-hFC vector was digested with AgeI and XhoI restriction enzymes, and the DNA sequence (CXCR2 1-48 aa, SEQ ID NO: 65) was cloned using the same restriction enzymes to obtain the CXCR2(NTD)-hFC recombinant plasmid.

[0156] Afterwards, hCXCR2 (1-48 aa) was inserted into the pET28a E. coli expression plasmid to produce the 6xHis-SUMO-CXCR2 (NTD) protein. Specifically, the pET28a-6xHis-SUMO vector was digested with SacI and XhoI restriction enzymes, and the DNA sequence (CXCR2 1-48 aa, SEQ ID NO: 66) and stop codon TAG were cloned using the same restriction enzymes to obtain the pET28a-His-Sumo-CXCR2 (NTD) recombinant plasmid.

[0157] Additionally, to construct IgG expression vectors, cDNAs encoding VL and VH with leader sequences were subcloned into the pdCMV vector between the HindIII-BsiWI and EcoRI-ApaI sites of the plasmid, respectively.

[0158] Specifically, the pdCMV-HC expression vector was digested with EcoRI and ApaI restriction enzymes, and the cDNA sequences of each IgG VH including the leader sequence (#1; SEQ ID NOs: 67, #18; SEQ ID NOs: 69, #54; SEQ ID NOs: 71, #56; SEQ ID NOs: 73) were cloned using the same restriction enzymes to obtain a recombinant plasmid. In addition, the pdCMV-LC expression vector was digested with HindIII and BsiWI restriction enzymes, and the cDNA sequences of each IgG VL including the leader sequence (#1; SEQ ID NOs: 68, #18; SEQ ID NOs: 70, #54; SEQ ID NOs: 72, #56; SEQ ID NOs: 74) were cloned using the same restriction enzymes to obtain a recombinant plasmid.

[0159]

[0160] Antibodies and reagents

[0161] The antibodies used in this study for flow cytometry analysis, immunoblotting, and immunostaining are listed in Table 1. Protein A agarose beads from Amicogen were used for antigen and IgG antibody purification. Ni-NTA agarose was used for antigen purification (QIAGEN). Geneticin was purchased from Gibco, and Calcein AM was purchased from Thermo Fisher Scientific. Recombinant human IL-8 / CXCL8 protein was obtained from R&D Systems, and SB220050 was purchased from Sigma-Aldrich. All chemicals were dissolved in appropriate reagents according to the manufacturers' instructions.

[0162] Antibody(source)SourceCat#CompanyUseAnti-human IgG conjugated Alexa Flour 647GoatA21445TermoFisherICC(1:200)Anti-human IgG conjugated Alexa Flour 594GoatA11014TermoFisherICC(1:200)Peroxidase AffiniPure Goat Anti-Human IgG(H+L)Goat109-035-088Jackson Immuno ResearchELISA(1:2500)Anti-human FC-HRPGoatab98624AbcamELISA(1:2500)Anti-HA-HRPRat12013819001RocheELISA(1:2500)Anti-phospho-p44 / 42 MAPKRabbit4370Cell Signaling TechnologyWB(1:2000)Anti-p44 / 42 MAPKRabbit4695Cell Signaling TechnologyWB(1:2000)Anti-Phospho-Akt (Ser473)Rabbit4060Cell Signaling TechnologyWB(1:2000)Anti-AKTRabbit4691Cell Signaling TechnologyWB(1:2000)PE anti-HA. 11 Epitope Tagmouse901517BioLegendICC(1:1500)

[0163] 아어: WB: western blot, ICC: immunocytochemistry

[0164]

[0165] 있조합 protein and full IgG antibody production

[0166] To purify the recombinant protein as an antigen, pcDNA3.1-CXCR2(NTD)-hFC was transfected into FreeStyleTM 293F (Thermo Fisher Scientific) cells, and the recombinant protein was harvested according to the manufacturer's instructions. For IgG antibody production, both heavy chain (HC) and light chain (LC) expression plasmids were cotransfected into 293F or ExpiCHO cells (Thermo Fisher Scientific). CXCR2(NTD)-hFC and IgG proteins were purified by affinity chromatography using protein A agarose beads. 6xHis-SUMO-CXCR2(NTD) was produced in an Escherichia coli expression system. Purification was performed by size exclusion chromatography using a protein purification system (Cytiva) followed by affinity chromatography using Ni-NTA agarose (QIAGEN).

[0167]

[0168] Biopanning using scFv antibody libraries based on phage display

[0169] The general procedure for phage display experiments was modified from that described in "Phage Display: A Laboratory Manual" [Barbas, CF, III; Burton, DR; Scott, JK, Silverman, GJ Eds. (2001) Phage Display: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York, 2001]. Briefly, human IgG or CXCR2(NTD)-hFC was coated onto immunotubes (MaxiSorp, Nunc) overnight at 4°C to remove hFC binders. Unbound antigen was washed away with phosphate-buffered saline (PBS). After blocking the surface with 3% skim milk in PBST, the scFv phage library was incubated with the coated CXCR2(NTD) in 3% skim milk at room temperature for 1 h. After washing with PBST to remove unbound phages, the captured phages were eluted by incubation with 100 mM triethanolamine for 10 minutes. The eluted phages were used to prepare phages for the next round of panning using the amplified ER2738 E. coli strain. After three rounds of panning, the output titers were measured, and single colonies from the final round were used for ELISA testing. Unique CDR sequences were selected from high-binding clones using the sequences obtained from scFv DNA sequencing and the IMGT V-quest (www.imgt.org / IMGTindex / V-QUEST.php) analysis program, and by ELISA analysis.

[0170]

[0171] scFv expression and extraction

[0172] A single colony expressing an individual scFv from the binder pool of the third round of biopanning was inoculated into 2x YT medium supplemented with carbenicillin (50 µg / ml) and cultured at 37°C for 6 h. To overexpress the scFv protein, 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the cell culture medium and the cells were further cultured overnight at 30°C. After removing the culture medium by centrifugation, the cell pellet was resuspended in 1x TES extraction solution containing 20% ​​w / v sucrose, 50 mM Tris, and 1 mM EDTA, pH 8.0. After incubation at 4°C for 30 min, 0.2x TES buffer was added to the suspension. After another 30 min incubation at 4°C, the periplasmic protein extract containing the scFv protein was harvested from the supernatant.

[0173]

[0174] ELISA

[0175] Antigens at 1 μg / ml or an appropriate concentration were dissolved in PBS and coated overnight on high-binding microplates (Corning 96-well half-area clear plates). After washing three times with PBS, the surfaces were coated with 3% skim milk in PBST (0.1% Tween-20) for 1 hour at room temperature. After coating, 30 μl of scFv extract or purified IgG and 3% skim milk solution were incubated with the coated antigens at 37°C for 1 hour. After washing three times with PBST, anti-HA-HRP or anti-human FC-HRP was incubated with 3% skim milk solution for 1 hour at 37°C. After an additional washing step, TMB substrate reagent (BD Biosciences) was added and incubated for 5 minutes before stopping with 1 N H2SO4. Individual optical density (OD) values ​​were measured at 450 nm using a microplate reader.

[0176]

[0177] Bio-layer Interferometry Kinetic Assay

[0178] Octet for biolayer interferometry (BLI) dynamic analysis ® The R4 protein analysis system (Sartorius) was used. His-tagged SUMO-CXCR2 (NTD) protein (150 nM) was coupled to the HIS1K biosensor. Candidate antibodies were prepared at seven concentrations (1.562 nM to 100 nM, half-diluted). The antigen-loaded sensors were immersed in the analyte wells for 120 s and then dissociated in the buffer wells for 180 s. Unloaded sensors served as reference sensors and were used to subtract baseline values. Data were generated using Octet Acquisition software and analyzed using Octet Analysis software. Kinetic parameters were estimated using a 1:1 binding model, and the corresponding KD, KON, and Koff values ​​were calculated.

[0179]

[0180] Flow cytometry

[0181] Cells were detached from the plate by brief exposure to cell dissociation buffer (Gibco) or 0.25% trypsin / EDTA. After washing with PBS, cells were blocked with 0.1% bovine serum albumin (BSA) in ice-cold PBS for 10 minutes. Cells were treated with appropriate concentrations of anti-CXCR2 candidates for 30 minutes at 4°C, followed by incubation with fluorescently conjugated secondary human IgG for 30 minutes at 4°C. After washing away nonspecific binding antibodies, fluorescence data were analyzed using NovoCyte Flow Cytometers (Agilent Technologies, Inc.).

[0182]

[0183] Immunoblotting

[0184] Cells were lysed with M-PER mammalian protein extraction reagent (Thermo Scientific) containing a protease inhibitor cocktail (Roche). The extracted proteins were reacted with 5X SDS-PAGE sample buffer for 1 hour at room temperature. The proteins were separated on an SDS-PAGE gel and then transferred to a nitrocellulose membrane. Bands were detected using a Chemidoc imaging system (Invitrogen).

[0185]

[0186] Calcium mobilization assay

[0187] Intracellular calcium concentration changes were measured using the Fluo-4 Direct Calcium Assay Kit according to the manufacturer's instructions (Invitrogen). Specifically, stable HEK293 cell lines expressing hCXCR2 were cultured in 96-well, clear-bottom black microplates (Greiner Bio-One). After serum starvation, cells were stained by incubating with Fluo-4 Direct Calcium Buffer containing candidate antibodies for 1 hour at 37°C in a 5% CO2 incubator. After treatment with CXCL8, fluorescence intensity was measured using a BioTek Synergy H1 microplate reader (Agilent USA).

[0188]

[0189] Cell migration assay

[0190] Cell migration was assessed using a Boyden chamber (8-μm pore size, Greiner Bio-one). A total of 1.5 × 10 5 AsPC-1 cells or 1X10 5Hey A8 cells were added to the upper chamber, and 600 μl of 1% FBS culture medium and 10 nM CXCL-8 were added to the lower chamber. Then, 20 μg / ml of candidate antibodies were added to the upper chamber. AsPC-1 or Hey A8 cells were incubated for 16 or 4 hours, respectively, and then stained with Calcein AM solution for 30 minutes. Cells that migrated to the underside of the transwell insert were captured under a fluorescence microscope, and the number of migrated cells was counted in five randomly selected fields.

[0191]

[0192] BrdU cell prolifaration assay

[0193] Cell proliferation was analyzed using the BrdU Cell Proliferation Assay Kit (Cell Signaling Technology). Specifically, AsPC-1 cells were cultured in RPMI medium containing 10% FBS in 96-well plates. The following day, cells were cultured in serum-free RPMI medium for 3 hours, after which candidate antibodies were added to the medium. After 1 hour of incubation, CXCL8 was added to the culture medium at a final concentration of 5% FBS. After 72 hours, cells were incubated with BrdU solution for 4 hours, and BrdU incorporation was detected according to the manufacturer's protocol. Individual optical density (OD) values ​​were measured at 450 nm using a microplate reader.

[0194]

[0195] Annexin-V apoptosis analysis

[0196] Apoptotic cells were analyzed using the Annexin V-FITC Apoptosis Detection Kit according to the manufacturer's instructions (Cell signaling Technology). Specifically, AsPC-1 cells were cultured in RPMI medium containing 10% FBS in 6-well plates. The following day, the cells were starved for approximately 1 hour with serum-free medium and then treated with 5% FBS RPMI medium containing CXCL8 or antibodies for 72 hours. The cells were harvested, washed twice with PBS, and incubated with Annexin V-FITC and PI in Annexin V binding buffer. The fluorescence of the stained cells was analyzed using a flow cytometer.

[0197]

[0198] Animals and xenograft

[0199] Six-week-old BALB / c nude female mice (18-20 g), free of specific pathogens, were purchased from a commercial animal breeder (COATECH, Gyeonggi-do, Korea). A total of 20 mice were randomly separated into polycarbonate cages, 5 per cage, and acclimated for 1 week in cages controlled at 20-24°C and 45-55% humidity. Mice were allowed free access to standard rodent food and water, and the cages were maintained on a 12-h light / dark cycle. After a 7-day acclimation period, a total of 5 × 10 6 AsPC-1 pancreatic carcinoma cells were injected subcutaneously into the right dorsal flank of nude mice. Twelve days after tumor induction, mice were examined for tumor size (80.19±29.06 mm 3 , range 37.42~127.32 mm 3) were divided into three groups according to the dose. The control group (4 mice) received intravenous saline injection, whereas the antibody-treated groups (IgG_18-treated group (5 mice) and IgG_56-treated group (5 mice)) received 10 mg / kg of each antibody (IgG_18 and IgG_56) intravenously once a week for 5 weeks. On the last treatment day, the mice were euthanized, and xenografts were harvested for further analysis. Tumor size and body weight were measured twice a week for 5 weeks and on the day of sacrifice. Subcutaneous tumor volumes were measured in two dimensions (length and width) using an Ultra-Cal VI caliper. Tumor growth inhibition (TGI) as a percentage of vehicle was calculated as the percentage difference between the daily average areas of the treatment groups over the same period. All experimental procedures were approved by the Institutional Animal Care and Use Committee of the Scripps Research Institute for the Antibody Research in Scripps Korea (Approval No. SIACUC-22-1-4).

[0200]

[0201] Test results

[0202] Screening of antibodies against human CXCR2 from a phage-displayed human library.

[0203] The N-terminal extracellular domain of human CXCR2 is known to be important for ligand binding and specificity [Gayle RB 3 rdet al., Importance of the amino terminus of the interleukin-8 receptor in ligand interactions. J Biol Chem. 1993 Apr 5;268(10):7283-9. PMID: 8463264.], [LaRosa GJet al., Amino terminus of the interleukin-8 receptor is a major determinant of receptor subtype specificity. J Biol Chem. 1992 Dec 15;267(35):25402-6. PMID: 1281158.], [Wu Let al., Discrete steps in binding and signaling of interleukin-8 with its receptor. J Biol Chem. 1996 Dec 6;271(49):31202-9], [Joseph PRet al., Probing the role of CXC motif in chemokine CXCL8 for high affinity binding and activation of CXCR1 and CXCR2 receptors. J Biol Chem. 2010 Sep 17;285(38):29262-9.], [Liu Ket al., Structural basis of CXC chemokine receptor 2 activation and signalling. Nature. 2020 Sep;585(7823):135-140.]. 또한, 핵자기 공명 및 결정학 분석 결과를 통한 세포외 루프 2도 CXCL8 결합의 중요성을 보여주었다 [Katancik JAet al., Mapping of the extracellular binding regions of the human interleukin-8 type B receptor. Biochem Biophys Res Commun. 1997 Mar 27;232(3):663-8.], [Ahuja SK et al., CXC chemokines bind to unique sets of selectivity determinants that can function independently and are broadly distributed on multiple domains of human interleukin-8 receptor B. Determinants of high affinity binding and receptor activation are distinct. J Biol Chem. 1996 Jan 5;271(1):225-32.], [Luo Z et al., Molecular modeling of interleukin-8 receptor beta and analysis of the receptor-ligand interaction. Protein Eng. 1997 Sep;10(9):1039-45.]. In this study, a 48 amino acid sequence located in the N-terminal domain (NTD) of hCXCR2 protein was selected for biopanning. First, a recombinant protein (CXCR2(NTD)-hFC) in which a human Fc fragment was fused to the C-terminus of CXCR2(NTD) was purified (Fig. 1a). In addition, SUMO-6XHis-CXCR2(NTD) recombinant protein was prepared to remove nonspecific binders or proteins that interact with the Fc fragment in the antigen (Fig. 1b). Phage display technology was performed primarily to screen for specific binders to the CXCR2(NTD)-Fc protein. In addition, the present inventors used a human library containing human synthetic single-chain variable fragments (scFv) for biopanning [Yang HY et al., Construction of a large synthetic human scFv library with six diversified CDRs and high functional diversity. Mol Cells.2009 Feb 28;27(2):225-35.]. After three rounds of biopanning, enzyme-linked immunosorbent assay (ELISA) was performed to initially select a group of binding antibodies, followed by sequence analysis to select nine unique scFv sequences.

[0204] In addition, we further confirmed the binding of scFv candidates to a stable HEK293 cell line expressing human CXCR2-GFP. As a result, using a biopanning procedure, we were able to select four unique scFv sequences (#1 (Table 2, SEQ ID NO: 29), #18 (Table 3, SEQ ID NO: 30), #54 (Table 4, SEQ ID NO: 31), and #56 (Table 5, SEQ ID NO: 31)) that bind to hCXCR2(NTD). Subsequently, the individual DNA sequences of the variable heavy (VH) and variable light (VL) chains were subcloned into an IgG expression vector, and full-length IgG proteins were produced (Fig. 2). ELISA was performed to confirm the interaction between hCXCR2(NTD) and full-length IgG candidates (#1, #18, #54, and #56), and it was found that the intensity increased in a dose-dependent manner (Figs. 3 and 4).

[0205] Furthermore, we precisely analyzed the direct affinity between CXCR2 (NTD) and candidate antibodies using a bio-layer interferometry (BLI)-based analysis. The individual KD values ​​of hCXCR2 (NTD) and IgG_18, IgG_54, and IgG_56 were found to be 8.9 nM, 250 nM, and 5.6 nM, respectively (Fig. 5). However, IgG_1 showed very weak binding compared to other candidate IgG proteins. In summary, we screened four human antibodies against hCXCR2 (NTD) by biopanning, and selected IgG_18 and IgG_56 antibodies as antibody candidates specifically targeting hCXCR2.

[0206] IgG_1 (#1)아미노산 서열서열번호VHFR1EVQLLESGGGLVQPGGSLRLSCAAS33CDR1GFTFSNYS1FR2MSWVRQAPGKGLEWVSW34CDR2IYYNGGST2FR3YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC35CDR3ARGPRICEIRKCYSANAMDV3FR4WGQGTLVTVSS36VLFR1QSVLTQPPSASGTPGQRVTISCSGS37CDR1SSNIGNNA4FR2VSWYQQLPGTAPKLLIY38CDR2ANS5FR3NRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC39CDR3GTWDASLSAYV6FR4FGGGTKLTVL40

[0207] IgG_18 (#18)아미노산 서열서열번호VHFR1EVQLLESGGGLVQPGGSLRLSCAAS41CDR1GFTFSNYA7FR2MSWVRQAPGKGLEWVSS42CDR2ISPSGSSK8FR3YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC43CDR3AKHNRHFDY9FR4WGQGTLVTVSS44VLFR1QSVLTQPPSASGTPGQRVTISCTGS45CDR1SSNIGSHY10FR2VSWYQQLPGTAPKLLIY46CDR2DNN11FR3HRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC47CDR3GSWDDSLNAYV12FR4FGGGTKLTVL48

[0208] IgG_54 (#54) amino acid SEQ ID NO:VHFR1EVQLLESGEGLVQPGGSLRLSCAAS49CDR1GFTFSNYS13FR2MSWVRQAPGKGLEWVSG50CDR2ISSGGGSK14FR3YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC51CDR3ARQATAFDY15FR4WGQGTLV TVSS52VLFR1QSVLTQPPSASGTPGQRVTISCTGS53CDR1SSNIGNNS16FR2VTWYQQLPGTAPKLLIY54CDR2SDS17FR3NRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC55CDR3GTWDDSLSAYV18FR4FCGGTKLTVL56

[0209] IgG_56 (#56) amino acid SEQ ID NO:VHFR1EVQLLESGGGLVQPGGSLRLSCAAS57CDR1GFTFSGYP19FR2MSWVRQAPGKGLEWVSS58CDR2ISSSGGNT20FR3YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC59CDR3ARFGIHHPDRALYYANAMDV21FR4W GQGTLVTVSS60VLFR1QSVLTQPPSASGTPGQRVTISCSGS61CDR1SSNIGNND22FR2VNWYQQLPGTAPKLLIY62CDR2ADN23FR3QRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYC63CDR3ATWDASLSAYV24FR4FGGGTKLTVL64

[0210]

[0211] Validation of antibody binding to various types of CXCR2-expressing cells

[0212] To determine whether the IgG_18 or IgG_56 antibody of the present invention has the ability to bind to hCXCR2-expressing cells, binding of the antibody to stable HEK293 cells expressing hCXCR2-GFP or hCXCR2-3xFLAG-3XHA was monitored using a flow cytometer.

[0213] Similar to the affinity measurement experimental results shown in Figures 3 to 5, FACS results showed that both IgG_18 and IgG_56 strongly bound to hCXCR2-expressing HEK293 cells (Figure 6). The N-terminal domain (1-48 aa) of hCXCR2 showed only 33% identity with the N-terminus of hCXCR1.

[0214] Additionally, antibody binding to HEK293 cells transiently expressing hCXCR1 or hCXCR2 was compared to determine antibody specificity. Both IgG18 and IgG56 of the present invention showed strong binding to cells expressing hCXCR2-GFP, but no significant binding to cells expressing hCXCR1-GFP (Fig. 7).

[0215] Next, we further analyzed the specificity of the candidate antibodies compared to other orthotropic CXCR2 proteins. Antibody binding to cells expressing mouse CXCR2 (mCXCR2-GFP) or rhesus macaque CXCR2 (rhCXCR2-GFP) was monitored. Neither IgG_18 nor IgG_56 exhibited any strong binding to mCXCR2-GFP-expressing cells (48% identity). Notably, the IgG_18 antibody was able to bind to rhCXCR2-GFP-expressing cells (76% identity), suggesting that it may share a similar epitope region with the hCXCR2 protein (Figure 7).

[0216] From these results, it can be seen that both IgG_18 and IgG_56 of the present invention have the ability to bind with high affinity to both CXCR2-recombinant proteins and CXCR2-expressing cells, and also have specificity for subtypes of chemokine receptors or heterologous CXCR2 proteins.

[0217]

[0218] CXCR2 antibodies inhibit CXCL8-induced signaling pathways

[0219] Chemokines that bind to G protein-coupled chemokine receptors mediate cell motility through changes in intracellular calcium concentrations [Rossi D et al., The biology of chemokines and their receptors. Annu Rev Immunol. 2000;18:217-42.]. It is well known that CXCR2 can increase intracellular calcium levels in the presence of agonists such as CXCL8. Therefore, calcium signals are a crucial and direct indicator of CXCR2 activation.

[0220] The inventors of the present invention predicted that if the anti-CXCR2 antibody according to the present invention binds to the CXCR2 receptor and blocks CXCL8 binding to the receptor, calcium elevation induced by CXCL8 treatment would be reduced. Accordingly, to assess whether both IgG_18 and IgG_56 of the present invention have functional properties as antagonistic antibodies in the CXCL8 / CXCR2 signaling axis, we first monitored CXCL8-mediated intracellular calcium elevation.

[0221] As expected, treatment of stable HEK293 cells expressing CXCR2 with 10 nM CXCL8 increased intracellular calcium levels, whereas CXCR2-expressing cells in the presence of IgG_18 or IgG_56 did not show any increase in intracellular calcium levels induced by CXCL8 (Fig. 8).

[0222] Meanwhile, CXCL8 / CXCR2 signaling is associated with tumor progression. CXCL8 / CXCR2 signaling is known to activate multiple signaling pathways, including mitogen-activated protein kinases (MAPK) or phosphatidylinositol-3 kinase (PI3K) / Akt pathways, and control numerous cellular activities related to cell proliferation, differentiation, motility, or apoptosis [Liu Q et al., The CXCL8-CXCR1 / 2 pathways in cancer. Cytokine Growth Factor Rev. 2016 Oct;31:61-71.], [Cheng Yet et al., Potential roles and targeted therapy of the CXCLs / CXCR2 axis in cancer and inflammatory diseases. Biochim Biophys Acta Rev Cancer. 2019 Apr;1871(2):289-312.].

[0223] To further verify the blocking effect of CXCL8 binding to CXCR2 antibodies, we monitored the activation of intracellular signaling mediators. Specifically, treatment of CXCR2 stable HEK293 cells with CXCL8 rapidly increased phosphorylation of p42 / 44 protein, whereas treatment of CXCR2 stable HEK293 cells with CXCL8 in the presence of IgG_18 or IgG_56 did not sufficiently increase p42 / 44 phosphorylation (Fig. 9). Consistent with the MAPK pathway, treatment with IgG_18 or IgG_56 was able to reduce the phosphorylation (activation) of AKT increased by CXCL8 treatment (Fig. 9).

[0224] From these results, it can be seen that both IgG_18 and IgG_56 of the present invention have an antagonistic function against CXCL8 / CXCR2 activation.

[0225]

[0226] CXCR2 inhibitory antibodies inhibit tumor formation

[0227] CXCL8 / CXCR2 signaling is important in cancer and inflammatory diseases. Several studies have reported that CXCR2 may play a role in chronic inflammation and in angiogenesis, tumorigenesis, and metastasis of colon, rectal, melanoma, lung, prostate, ovarian, and pancreatic cancers. [Stadtmann A et al., CXCR2: From Bench to Bedside. Front Immunol. 2012 Aug 24;3:263.], [Gabellini C et al., Functional activity of CXCL8 receptors, CXCR1, and CXCR2, on human malignant melanoma progression. Eur J Cancer. 2009 Sep;45(14):2618-27.], [Ohri CM et al., Chemokine receptor expression in tumor islets and stroma in non-small cell lung cancer. BMC Cancer. 2010 Apr 29;10:172.], [Reiland Jet al., CXC-chemokines stimulate invasion and chemotaxis in prostate carcinoma cells through the CXCR2 receptor. Prostate. 1999 Oct 1;41(2):78-88.], [Wang Bet al., A growth-related oncogene / CXC chemokine receptor 2 autocrine loop contributes to cellular proliferation in esophageal cancer. Cancer Res. 2006 Mar 15;66(6):3071-7.], [Yang et al., CXCR2 promotes ovarian cancer growth through dysregulated cell cycle, diminished apoptosis, and enhanced angiogenesis. Clin Cancer Res. 2010 Aug 1;16(15):3875-86.], [Li A et al., CXCR2-Dependent Endothelial Progenitor Cell Mobilization in Pancreatic Cancer Growth. Transl Oncol. 2011 Feb 1;4(1):20-8.]. Overexpression of CXCR2 has been reported to be highly expressed not only in immune cells such as neutrophils but also in many primary cancer cells. Through Fig. 6, it was confirmed that the IgG_18 and IgG_56 antibodies according to the present invention specifically bind to hCXCR2-expressing HEK293T cells.

[0228] To monitor whether CXCR2 is expressed in various cancer cell lines and whether CXCR2 antibodies can bind to them, immunostaining and flow cytometry analysis were performed. The analysis results confirmed that both IgG18 and IgG56 according to the present invention strongly bound to various cancer cell lines, including pancreatic, ovarian, colon, and lung cancer cell lines (Figure 10). This supports the finding that CXCR2 is expressed in many cancer cells and that CXCR2 signaling may play a critical role in tumorigenesis.

[0229] Furthermore, to explore the potential of IgG_18 and IgG_56 according to the present invention as inhibitory antibodies for cancer treatment, we first examined their effects on cell proliferation in pancreatic cancer cell lines. As expected, CXCL8 significantly promoted AsPC-1 cell proliferation in a dose-dependent manner, as confirmed by a BrdU cell proliferation assay (Fig. 11). Furthermore, this cancer cell proliferation was not significantly increased in the presence of IgG_18 or IgG_56, even in the 30 nM CXCL8-treated group.

[0230] Next, we sought to further analyze whether CXCR2 antibodies have a functional antagonistic effect on cancer cell migration. As a result, a Transwell cell migration assay revealed that AsPC-1 cells significantly migrated toward the lower chamber toward high concentrations of CXCL8, whereas treatment with IgG_18 or IgG_56 of the present invention significantly reduced the increase in the number of AsPC-1 cells transmembrane mediated by CXCL8 (Fig. 12).

[0231] Additionally, to further evaluate the antagonistic effect of the antibodies on cancer cells, the level of apoptosis was monitored. Cancer cell death was detected using flow cytometry after Annexin V and propidium iodide (PI) staining. As shown in Figure 13, when AsPC-1 cells were treated with CXCL8, cancer cell death was reduced compared to vehicle-treated cells, whereas when treated with IgG18 or IgG56 of the present invention, the decreased cancer cell death in response to CXCL8 was increased. These results indicate that CXCR2 blockade by the IgG18 or IgG56 antibodies of the present invention induces apoptosis in AsPC-1 pancreatic cancer cells.

[0232] In summary, IgG18 and IgG56 according to the present invention can bind to various cancer cells including pancreatic cancer cells and have an anti-tumor effect by inhibiting the CXCL8 / CXCR2 signal transduction pathway in cancer.

[0233]

[0234] CXCR2 antibodies mitigate tumor growth in xenograft mouse model systems.

[0235] To evaluate the potential therapeutic benefits of the antibody according to the present invention, we next investigated its antitumor effect on tumors in vivo. A xenograft mouse model in which AsPC-1 pancreatic carcinoma was subcutaneously injected into BALB / c nude mice was used. The tumor was 80 mm in size. 3 After allowing the tumors to grow until they reached , the mice were administered 10 mg / kg of IgG_18 or IgG_56 (intravenous injection). As shown in Fig. 14A-C, the tumor volume and size, as well as the final tumor weight, were significantly reduced compared to the mice injected with PBS, and in particular, the mice treated with 10 mg / kg of IgG_18 showed a stronger effect in suppressing the increase in tumor weight (Fig. 14C). In addition, the body weights of the tested mice were not affected by the administration of the candidate antibodies, indicating that the antibodies had no noticeable side effects in the in vivo experiment (Fig. 14D).

[0236] Taken together, these results suggest that IgG_18 and IgG_56 exhibit antitumor effects by inhibiting CXCR2 signaling in a pancreatic cancer cell xenograft model.

[0237]

[0238] From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as encompassing all changes or modifications derived from the meaning and scope of the following claims and their equivalent concepts, rather than the detailed description above.

Claims

1. An anti-CXCR2 antibody or antigen-binding fragment thereof comprising any one of the heavy chain variable region and light chain variable region selected from 1) and 2) below: 1) a heavy chain variable region comprising a CDR1 region consisting of an amino acid sequence of SEQ ID NO: 7, a CDR2 region consisting of an amino acid sequence of SEQ ID NO: 8, and a CDR3 region consisting of an amino acid sequence of SEQ ID NO: 9, and a light chain variable region comprising a CDR1 region consisting of an amino acid sequence of SEQ ID NO: 10, a CDR2 region consisting of an amino acid sequence of SEQ ID NO: 11, and a CDR3 region consisting of an amino acid sequence of SEQ ID NO: 12; and 2) A heavy chain variable region comprising a CDR1 region consisting of the amino acid sequence of SEQ ID NO: 19, a CDR2 region consisting of the amino acid sequence of SEQ ID NO: 20, and a CDR3 region consisting of the amino acid sequence of SEQ ID NO: 21, and a light chain variable region comprising a CDR1 region consisting of the amino acid sequence of SEQ ID NO: 22, a CDR2 region consisting of the amino acid sequence of SEQ ID NO: 23, and a CDR3 region consisting of the amino acid sequence of SEQ ID NO:

24.

2. In paragraph 1, the anti-CXCR2 antibody or antigen-binding fragment thereof, 1) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 25 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 26; or 2) An anti-CXCR2 antibody or antigen-binding fragment thereof, characterized by comprising a heavy chain variable region consisting of an amino acid sequence of SEQ ID NO: 27 and a light chain variable region consisting of an amino acid sequence of SEQ ID NO:

28.

3. In paragraph 1, An anti-CXCR2 antibody or antigen-binding fragment thereof, characterized in that the anti-CXCR2 antibody or antigen-binding fragment thereof has an activity of inhibiting the growth or metastasis of cancer cells expressing the CXCR2 protein and inducing apoptosis.

4. In paragraph 1, The above anti-CXCR2 antibody or antigen-binding fragment thereof is an anti-CXCR2 antibody or antigen-binding fragment thereof, characterized in that it blocks the binding of CXCL8 (CXC motif chemokine ligand 8) and CXCR2.

5. In paragraph 1, An anti-CXCR2 antibody or an antigen-binding fragment thereof, characterized in that the above anti-CXCR2 antibody is a monoclonal antibody.

6. In paragraph 1, An anti-CXCR2 antibody or an antigen-binding fragment thereof, characterized in that the antigen-binding fragment is any one selected from Fab, Fab', F(ab')2, scFv, Fv, dsFv, diabody, Fd and Fd'.

7. A polynucleotide encoding an anti-CXCR2 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6.

8. In paragraph 7, A polynucleotide characterized in that the polynucleotide comprises any one sequence selected from SEQ ID NO: 30 and SEQ ID NO:

32.

9. An expression vector comprising the polynucleotide of clause 7.

10. A transformant transformed with the expression vector of Article 9.

11. A pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient an anti-CXCR2 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6, a polynucleotide encoding the anti-CXCR2 antibody or an antigen-binding fragment thereof, or an expression vector comprising the polynucleotide.

12. In paragraph 11, A composition characterized in that the cancer is at least one selected from esophageal cancer, stomach cancer, colon cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, rectal cancer, melanoma, breast cancer, cervical cancer, endometrial cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, soft tissue sarcoma, lymphoma, and multiple myeloma blood cancer.

13. A composition for diagnosing CXCR2 overexpressing cancer, comprising an anti-CXCR2 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6, a polynucleotide encoding the anti-CXCR2 antibody or an antigen-binding fragment thereof, or an expression vector comprising the polynucleotide.

14. A diagnostic kit for CXCR2 overexpressing cancer comprising the composition and instructions of Article 13. 15.(a) A step of contacting an anti-CXCR2 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6, a polynucleotide encoding the anti-CXCR2 antibody or an antigen-binding fragment thereof, or an expression vector comprising the polynucleotide, with a biological sample isolated from an individual suspected of having CXCR2 overexpressing cancer; (b) a step of measuring the expression level of CXCR2 protein from the biological sample; and (c) a step of determining that the cancer is CXCR2 overexpression cancer if the expression level of the CXCR2 protein measured in the step (b) is higher than that of the control group; a method for providing information necessary for diagnosing CXCR2 overexpression cancer.

16. A method for treating cancer, comprising administering to a subject an anti-CXCR2 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 6, a polynucleotide encoding the anti-CXCR2 antibody or an antigen-binding fragment thereof, or an expression vector comprising the polynucleotide.

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