KR20250133491A - Novel Antibody Specific for human CCL7 and Uses Thereof - Google Patents

Abstract

The present invention relates to a novel antibody that binds to human CCL7 (C-C motif Chemokine ligand 7). The antibody of the present invention is an antibody that has the effect of inhibiting chemotaxis of monocytes, and can be developed as a therapeutic agent for diseases that overexpress CCL7 protein, and can also be utilized as a diagnostic system for CCL7 protein.

Description

{Novel Antibody Specific for human CCL7 and Uses Thereof}

The present invention relates to a novel antibody binding to human CCL7 (C-C motif Chemokine ligand 7) and its use.

CCL7 (C-C motif Chemokine ligand 7) is a cytokine and a small protein belonging to the CC chemokine family. CCL7 is expressed on various cell types, including stromal cells, keratinocytes, airway smooth muscle cells, parenchymal cells, fibroblasts, and leukocytes, as well as tumor cells. CCL7 acts primarily as a chemoattractant for several leukocytes, including monocytes, eosinophils, basophils, dendritic cells, neutrophils, NK cells, and activated T lymphocytes, and thus, as a chemotactic factor, CCL7 plays a role in mediating the immune response by recruiting leukocytes to infected tissues.

Many diseases have been reported to be associated with CCL7 dysregulation. For example, abnormal increases in CCL7 exacerbate many diseases, such as HIV and lesional psoriasis. Furthermore, CCL7 is associated with various immune diseases, including ulcerative colitis, multiple sclerosis, non-atopic asthma, and atopic asthma. Studies have shown that its expression is elevated in renal, gastric, and colon cancers. Furthermore, studies have shown that CCL7 expression is involved in cancer cell metastasis and immune cell infiltration not only in colon cancer but also in major intractable cancers, such as lung, renal, and breast cancers, inducing changes in the tumor microenvironment and promoting tumor invasion and metastasis. High CCL7 expression has been observed in liver metastases from patients with metastatic colon cancer, and this expression has been confirmed to be secreted by tumor-associated macrophages (TAMs) in the metastatic tumors.

Chemotherapy for colorectal cancer patients primarily involves first-generation chemotherapy, such as FOLFOX (5-Fluorouarcil + Leucovorin + Oxaliplatin) or FOLFIRI (5-Fluorouarcil + Leucovorin + Irinotecan). For patients with metastatic colorectal cancer, combinations of the aforementioned chemotherapy agents and targeted anticancer agents are used to enhance treatment efficacy. However, these treatments are ineffective, and up to 60% of patients die.

When existing targeted anticancer drug-based treatments are ineffective, immunotherapy, which has been extensively studied recently, can be used. However, it is effective in less than 40% of patients with microsatellite instability (MSI) colorectal cancer. Furthermore, in microsatellite stable (MSS) colorectal cancer, which accounts for more than 80% of colorectal cancer cases, the response rate to immunotherapy is 0%, making it inapplicable. Intensive research is being conducted to develop adjuvant or complementary therapies that can be used in combination with immunotherapy to elicit the effects of immunotherapy in MSS colorectal cancer, where immunotherapy is completely ineffective.

The novel human CCL7 binding antibody has a mechanism of inhibiting the growth and metastasis of cancer cells by neutralizing CCL7. It is expected that the development of a CCL7-targeting antibody treatment will increase the treatment efficacy when used in combination with existing drugs in patients who do not respond to existing treatments.

Accordingly, the inventors of the present invention discovered a novel human antibody that binds to the CCL7 protein through human synthetic antibody phage display library panning, confirmed its binding ability to the CCL7 protein, and completed the present invention by confirming that it has an effect of inhibiting the chemotaxis of human monocytes induced by CCL7.

KR 10-1238196 B1 KR 10-2017-0044050 A1

An object of the present invention is to provide an antibody or an antigen-binding fragment thereof that specifically binds to CCL7.

Another object of the present invention is to provide a nucleic acid encoding an antibody or an antigen-binding fragment thereof.

Another object of the present invention is to provide a vector comprising the nucleic acid.

Another object of the present invention is to provide a host cell comprising the above vector.

Another object of the present invention is to provide a method for producing an antibody or antigen-binding fragment thereof that specifically binds to CCL7.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer, comprising the antibody or an antigen-binding fragment thereof as an active ingredient.

Another object of the present invention is to provide a composition for diagnosing cancer comprising the antibody or an antigen-binding fragment thereof as an active ingredient.

The present invention provides an antibody or antigen-binding fragment thereof that specifically binds to CCL7.

The present invention also provides a nucleic acid encoding an antibody or an antigen-binding fragment thereof.

In addition, the present invention provides a vector comprising the nucleic acid.

Additionally, the present invention provides a host cell comprising the vector.

Additionally, the present invention provides a method for producing an antibody or antigen-binding fragment thereof that specifically binds to CCL7.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the antibody or an antigen-binding fragment thereof as an active ingredient.

In addition, the present invention provides a composition for diagnosing cancer comprising the antibody or an antigen-binding fragment thereof as an active ingredient.

The present invention relates to a novel antibody that binds to human CCL7 (C-C motif Chemokine ligand 7). The antibody of the present invention is an antibody that has the effect of inhibiting chemotaxis of monocytes, and can be developed as a therapeutic agent for diseases that overexpress CCL7 protein, and can also be utilized as a diagnostic system for CCL7 protein.

Figure 1 is a flow chart of a method for adding histones in a method for producing a humanized antibody specific for the CCL7 antigen according to Example 1 of the present invention.
FIG. 2 is a diagram showing the results of measuring the number of phages eluted each time in the first panning according to FIG. 1 of the present invention.
Figure 3 is a flow chart of a method for applying BSA as a blocking agent in a method for producing a humanized antibody specific for the CCL7 antigen according to Example 1 of the present invention.
FIG. 4 is a diagram showing the results of measuring the number of phages eluted each time in the second panning according to FIG. 3 of the present invention.
Figure 5 is a diagram showing the phage ELISA results of 1D12, an antibody selected through the panning of Figure 2.
Figure 6 is a diagram showing the phage ELISA results of 1C1, an antibody selected through the panning of Figure 3.
Figure 7 is a diagram showing the results of purification of two types of human CCL7 binding novel antibodies, 1D12 and 1C1, confirmed through SDS-PAGE.
Figure 8 is a diagram showing the results of confirming the purity of the 1D12 antibody through SE-HPLC analysis.
Figure 9 is a diagram showing the results of confirming the purity of the 1C1 antibody through SE-HPLC analysis.
Figure 10 is a graph showing the results of confirming the binding ability of two types of human CCL7 binding novel antibodies, 1D12 and 1C1, to the CCL7 antigen.
Figure 11 is a graph showing the results of confirming the affinity of two types of human CCL7 binding novel antibodies, 1D12 and 1C1, for the CCL7 antigen.
Figure 12 is a graph showing the results of confirming whether the novel human CCL7-binding antibody 1D12 can inhibit chemotaxis of human monocytes by CCL7.
Figure 13 is a graph showing the results of calculating the results of Figure 9 as fold change.
Figure 14 is a graph showing the results of confirming the cancer cell proliferation rate according to human recombinant CCL7 treatment.
Figure 15 is a graph showing the results of the first experiment confirming the growth inhibitory effect of MAB282 antibody (positive control) and human CCL7 binding novel antibody 1D12 on cancer cells.
Figure 16 is a graph showing the results of a second experiment confirming the growth inhibitory effect of MAB282 antibody (positive control) and two types of human CCL7-binding novel antibodies, 1D12 and 1C1, on cancer cells.

The present invention is described in detail below.

The present invention provides an antibody or antigen-binding fragment thereof that specifically binds to CCL7.

In the present invention, the antibody is also called immunoglobulin (Ig), and is a general term for proteins that selectively act on antigens and participate in biological immunity. Whole antibodies found in nature are generally composed of two pairs of light chains (LC) and heavy chains (HC), which are polypeptides composed of multiple domains, or have a structure of two pairs of HC/LC as their basic units. There are five types of heavy chains that make up mammalian antibodies, indicated by the Greek letters α, δ, ε, γ, and μ, and depending on the type of heavy chain, they make up different types of antibodies, such as IgA, IgD, IgE, IgG, and IgM. There are two types of light chains that make up mammalian antibodies, indicated by λ and κ.

The heavy and light chains of antibodies are structurally divided into variable and constant regions according to the variability of the amino acid sequence. The constant region of the heavy chain is composed of three or four heavy chain constant regions, such as CH1, CH2, and CH3 (IgA, IgD, and IgG antibodies) and CH4 (IgE and IgM antibodies), depending on the type of antibody, and the light chain is composed of one constant region, CL. The variable regions of the heavy and light chains are each composed of one domain, the heavy chain variable region (VH) or the light chain variable region (VL). The light and heavy chains are linked by a single covalent disulfide bond with their variable and constant regions aligned side by side, and the heavy chains of the two molecules bound to the light chain are linked by two covalent disulfide bonds to form the entire antibody. Whole antibodies specifically bind to antigens through the variable regions of the heavy and light chains, and since whole antibodies are composed of two pairs of heavy and light chains (HC/LC), one molecule of whole antibodies has bivalent monospecificity, binding to the same two antigens through the two variable regions.

The variable region, which includes the antigen-binding site of an antibody, is subdivided into a framework region (FR) with low sequence variability and a complementary determining region (CDR), which is a hypervariable region with high sequence variability. The CDR, which has the highest sequence variability within the variable region of an antibody, is the site that directly binds to the antigen and is most important for the antigen specificity of the antibody.

The present inventors developed antibodies and fragments thereof that specifically bind to the CCL7 protein, and confirmed that these antibodies specifically bind only to the CCL7 protein through the unique variable region CDR sequence configuration they possess.

Specifically, the antibody or fragment thereof of the present invention is characterized by comprising a light chain variable region (VL) and a heavy chain variable region (VH) comprising the following unique CDR sequences:

An antibody or antigen-binding fragment thereof comprising a light chain variable region comprising a light chain CDR1 defined by the amino acid sequence of SEQ ID NO: 1, a light chain CDR2 defined by the amino acid sequence of SEQ ID NO: 2, and a light chain CDR3 defined by the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising a heavy chain CDR1 defined by the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 defined by the amino acid sequence of SEQ ID NO: 5, and a heavy chain CDR3 defined by the amino acid sequence of SEQ ID NO: 6; and may comprise a light chain variable region having the amino acid sequence of SEQ ID NO: 13 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 14.

An antibody or antigen-binding fragment thereof comprising a light chain variable region comprising a light chain CDR1 defined by the amino acid sequence of SEQ ID NO: 7, a light chain CDR2 defined by the amino acid sequence of SEQ ID NO: 8, and a light chain CDR3 defined by the amino acid sequence of SEQ ID NO: 9; and a light chain variable region comprising a heavy chain CDR1 defined by the amino acid sequence of SEQ ID NO: 10, a heavy chain CDR2 defined by the amino acid sequence of SEQ ID NO: 11, and a heavy chain CDR3 defined by the amino acid sequence of SEQ ID NO: 12; and may comprise a light chain variable region having the amino acid sequence of SEQ ID NO: 29 and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 30.

The antibody that specifically binds to the human CCL7 protein according to the present invention is not limited in type, as long as it has a composition of the above-described combination of CDRs. Specifically, it may be selected from the group consisting of IgG, IgA, IgM, IgE, and IgD, and an IgG antibody is particularly preferred.

The antibody of the present invention may include all forms of chimeric antibodies, humanized antibodies, human antibodies, etc., as long as they contain the unique CDR configuration described above, and may preferably be a human antibody.

In addition, in the present invention, an antibody fragment means an antibody fragment that maintains the antigen-specific binding ability of the entire antibody, and specifically, may be in the form of a diabody, Fab, Fab′, F(ab)2, F(ab′)2, Fv, and scFv.

Fab (fragment antigen-binding) is an antigen-binding fragment of an antibody, consisting of one variable domain and one constant domain of each heavy chain and light chain. F(ab')2 is a fragment produced by hydrolyzing an antibody with pepsin, and has a structure in which two Fabs are linked by a disulfide bond at the heavy chain hinge. F(ab') is a monomeric antibody fragment in which a heavy chain hinge is added to Fab, which is obtained by reducing the disulfide bond of the F(ab')2 fragment. Fv (variable fragment) is an antibody fragment composed only of the variable domains of each heavy chain and light chain. ScFv (single chain variable fragment) is a recombinant antibody fragment in which the heavy chain variable region (VH) and the light chain variable region (VL) are linked by a flexible peptide linker. Diabody refers to a fragment in which the VH and VL of scFv are connected by a very short linker and do not bind to each other, but bind to the VL and VH of another scFv of the same type, forming a dimer.

The antibodies or fragments thereof of the present invention can be produced using methods known in the art, for example, phage display methods or yeast cell surface expression systems. Methods for producing scFvs include those described in U.S. Patent Nos. 4,946,778 and 5,258,498, and methods for recombinantly producing Fab, Fab', and F(ab')2 fragments include those described in WO 92/22324.

The present invention also provides a nucleic acid encoding an antibody or an antigen-binding fragment thereof.

The term 'nucleic acid' in the present invention may be described as an oligonucleotide or a polynucleotide, and includes DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA produced using nucleotide analogs (e.g., peptide nucleic acids and non-naturally occurring nucleotide analogs), and hybrids thereof. The nucleic acid may be single-stranded or double-stranded.

The nucleic acid of the present invention is not particularly limited in its sequence as long as it encodes the antibody of the present invention or an antigen-binding fragment thereof as described above. The nucleic acid encoding the antibody of the present invention or an antigen-binding fragment thereof as described above can be obtained by a method well known in the art. For example, codons can be analyzed based on the amino acid sequence corresponding to the full-length sequence of the antibody of the present invention as described above, or a portion of the heavy chain and light chain (particularly, a region including the heavy chain variable region and the light chain variable region), and mRNA or cDNA can be constructed through these codon analyses. Such nucleic acids can be synthesized using an oligonucleotide synthesis technique known in the art, such as the polymerase chain reaction (PCR) method.

The antibody or antigen-binding fragment thereof of the present invention may comprise a light chain nucleic acid sequence comprising a light chain CDR1 nucleic acid sequence of SEQ ID NO: 7, a light chain CDR2 nucleic acid sequence of SEQ ID NO: 8, and a light chain CDR3 nucleic acid sequence of SEQ ID NO: 9, and a heavy chain nucleic acid sequence comprising a heavy chain CDR1 nucleic acid sequence of SEQ ID NO: 10, a heavy chain CDR2 nucleic acid sequence of SEQ ID NO: 11, and a heavy chain CDR3 nucleic acid sequence of SEQ ID NO: 12, and a light chain nucleic acid sequence comprising a light chain CDR1 nucleic acid sequence of SEQ ID NO: 23, a light chain CDR2 nucleic acid sequence of SEQ ID NO: 24, and a light chain CDR3 nucleic acid sequence of SEQ ID NO: 25, and a heavy chain CDR1 nucleic acid sequence of SEQ ID NO: 26, a heavy chain CDR2 nucleic acid sequence of SEQ ID NO: 27, and a heavy chain CDR3 nucleic acid sequence of SEQ ID NO: 28.

In addition, the present invention provides a vector comprising the nucleic acid.

The above vector is a recombinant expression vector, and in the present invention, 'recombinant' can be used interchangeably with 'genetic manipulation', and means producing a gene that does not exist in nature by using molecular cloning experimental techniques such as modifying, cutting, and connecting genes.

In the present invention, expression means that a protein or nucleic acid is produced in a cell.

In the present invention, the term "recombinant expression vector" refers to a vector capable of expressing a desired protein or nucleic acid (RNA) in a suitable host cell, and refers to a genetic construct that includes essential regulatory elements operably linked to enable expression of a polynucleotide (gene) insert. "Operably linked" means that a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein or RNA are functionally linked to perform a general function, and that a gene is linked so that it can be expressed by the expression control sequence. The term "expression control sequence" refers to a DNA sequence that controls the expression of a polynucleotide sequence operably linked in a specific host cell. Such control sequences include a promoter for initiating transcription, an arbitrary operator sequence for regulating transcription, a sequence encoding a suitable mRNA ribosome binding site, a sequence regulating the termination of transcription and translation, an initiation codon, a stop codon, a polyadenylation signal, an enhancer, and the like. Therefore, the recombinant expression vector according to the present invention means a genetic construct in which a nucleic acid encoding the antibody of the present invention having a unique CDR configuration capable of specifically binding to human CCL7 protein as described above is operably linked so that it can be expressed in an appropriate host cell.

The recombinant expression vector of the present invention is not particularly limited in type as long as it is a vector commonly used in the cloning field, and examples thereof include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, and viral vectors. The plasmids include plasmids derived from Escherichia coli (pBR322, pBR325, pUC118 and pUC119, pET-22b(+)), plasmids derived from Bacillus subtilis (pUB110 and pTP5), and plasmids derived from yeast (YEp13, YEp24, and YCp50), and the viruses may include animal viruses such as retrovirus, adenovirus, or vaccinia virus, insect viruses such as baculovirus, and pcDNA, etc.

A plasmid, a type of vector, is a linear or circular double-stranded DNA molecule into which foreign polynucleotide fragments can be ligated. Another type of vector is a viral vector (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), in which additional DNA fragments can be introduced into the viral genome. Certain vectors are capable of autonomous replication within a host cell into which they are introduced (e.g., bacterial vectors, including those of bacterial origin and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell and are thereby replicated together with the host genome.

The nucleic acids encoding the heavy chain and light chain of the antibody according to the present invention, or fragments thereof (particularly fragments including the heavy chain variable region and the light chain variable region), may be contained in separate recombinant expression vectors, or may be contained in one recombinant expression vector.

The present invention also provides a host cell comprising the vector. That is, the present invention provides a cell transformed with a recombinant expression vector comprising a nucleic acid encoding an antibody or antigen-binding fragment thereof according to the present invention.

The type of the cell (host cell) of the present invention is not particularly limited as long as it is a cell that can be used to express a nucleic acid encoding an antibody or an antigen-binding fragment thereof included in the recombinant expression vector of the present invention. The cell (host cell) transformed with the recombinant expression vector according to the present invention may be a prokaryote (e.g., Escherichia coli), a eukaryote (e.g., yeast or other fungi), a plant cell (e.g., tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, an insect cell, or a hybridoma derived therefrom). Preferably, the cell may be derived from a mammal including a human.

Examples of useful mammalian host cells include monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651), human embryonic kidney line (293 or 293 cells subcloned from suspension culture [Graham et al., 1977, J Gen Virol. 36: 59]), baby hamster kidney cells (BHK, ATCC CCL10), Chinese hamster ovary cells/-DHFR" (CHO, Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77: 4216; e.g., DG44), mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod. 23:243-251), monkey kidney cells (CV1 ATCC CCL 70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical cancer cells. (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human hepatocytes (Hep G2, HB 8065), mouse breast tumor (MMT 060562, ATCC CCL51), TRI cells (Mather et al., 1982, Annals NY. Acad. Sci. 383: 44-68), MRC 5 cells, FS4 cells, human hepatoma cell line (Hep G2), HEK 293 cells (human embryonic kidney cells) and Expi293 cells, preferably Expi293 cells.

The cell provided in the present invention is a cultured cell that can be transformed or transfected with the nucleic acid of the present invention or a vector containing the same, and which can subsequently be expressed within the host cell. A recombinant cell refers to a cell transformed or transfected with a polynucleotide to be expressed.

The medium composition, culture conditions, culture time, etc. for culturing the above cells can be appropriately selected according to methods commonly used in the art. Commercially available media, such as Ham's F1O (Sigma-Aldrich Co., St. Louis, MO), minimum essential medium (MEM, Sigma-Aldrich Co.), RPMI-1640 (Sigma-Aldrich Co.), and Dulbecco's Eagle's medium (DMEM, Sigma-Aldrich Co.), are suitable for culturing cells. If necessary, hormones and/or other growth factors, salts, buffers, nucleotides, antibiotics, trace elements, and glucose or an equivalent energy source can be added to the media.

In addition, the present invention provides a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to CCL7, comprising the steps of culturing a host cell; and recovering the antibody or an antigen-binding fragment thereof from the cultured cell.

As described above, since the antibody of the present invention or its antigen-binding fragment has a specific amino acid sequence of a region (CDR and variable region including the same) important for antigen recognition and binding, a person skilled in the art can easily and repeatedly mass-produce the antibody of the present invention based on this.

The above culture may vary in medium composition and culture conditions depending on the type of cell, and the conditions for expression of polynucleotides depending on the cell can be appropriately selected and controlled by a person skilled in the art, and the above-mentioned examples can be referenced with respect to the culture medium of the cells of the present invention.

The antibody molecule may be accumulated in the cytoplasm of a cell, secreted from the cell, or targeted to the periplasm or the extracellular medium by an appropriate signal sequence, and it is preferred that it be targeted to the periplasm or the extracellular medium. In addition, it is preferred that the produced antibody molecule be refolded and made to have a functional conformation using a method well known to those skilled in the art. In addition, when producing an IgG type antibody, the heavy chain and light chain may be expressed in separate cells and produced to form a complete antibody by contacting the heavy chain and the light chain in a separate step, or the heavy chain and the light chain may be expressed in the same cell to form a complete antibody inside the cell.

The recovery of the antibody or antigen-binding fragment thereof can be appropriately selected and controlled by a person skilled in the art, taking into consideration the characteristics of the produced antibody or its fragment polypeptide, the characteristics of the host cell, the expression method, or whether the polypeptide is targeted, etc. For example, an antibody or fragment thereof secreted into a culture medium can be recovered by obtaining a medium in which host cells are cultured and removing impurities through a filter (such as ultrafiltration) or centrifugation. If necessary, cells may be lysed within a range that does not affect the functional structure of the antibody or its fragment to release and recover antibodies present in specific organelles or cytoplasm within the cell. In addition, the obtained antibody may be further subjected to a process of further removing impurities and concentrating them through a method such as chromatography, filtration using a filter, etc., or dialysis. A protease inhibitor, such as PMSF, may be included in any preceding step to suppress proteolysis, and antibiotics may be included to prevent the growth of accidental contaminants.

Antibodies prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography.

In addition, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising the antibody or an antigen-binding fragment thereof as an active ingredient.

In addition, the antibody of the present invention or an antigen-binding fragment thereof has an effect of preventing or treating cancer by inhibiting the growth of cancer. Therefore, the present invention provides a pharmaceutical composition for preventing and treating cancer comprising the antibody of the present invention or an antigen-binding fragment thereof as an active ingredient. The antibody of the present invention or a pharmaceutical composition comprising the same inhibits the growth of tumor cells, and thus can be applied to various cancers, and is not limited thereto, for example, the cancers include colon cancer, lung cancer, liver cancer, stomach cancer, esophageal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, cervical cancer, endometrial cancer, choriocarcinoma, ovarian cancer, breast cancer, thyroid cancer, brain cancer, head and neck cancer, skin cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, colon cancer, bone cancer, brain tumor, thyroid cancer, leukemia, lymphoma, and multiple myeloma.

It may be selected from a group consisting of bone marrow and blood cancers, preferably cancers that specifically express CCL7.

The pharmaceutical composition according to the present invention may comprise the antibody or fragment thereof alone, or may additionally comprise one or more pharmaceutically acceptable carriers. The term "pharmaceutically effective amount" as used herein refers to an amount that exhibits a greater response than a negative control, and preferably refers to an amount sufficient to treat cancer.

As used herein, “pharmaceutically acceptable” refers to a non-toxic composition that is physiologically acceptable and does not inhibit the action of the active ingredient when administered to humans and does not typically cause allergic reactions such as gastrointestinal upset, dizziness, or similar reactions.

In the pharmaceutical composition according to the present invention, the antibody or fragment thereof can be administered in various oral and parenteral dosage forms during clinical administration, and when formulated, it can be prepared using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

For example, it can be formulated as an injection for parenteral administration. In the case of the injection, it must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. Examples of suitable carriers for the injection include, but are not limited to, solvents or dispersion media including water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), mixtures thereof, and/or vegetable oils. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% dextrose. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal can be additionally included. Additionally, the above injections may in most cases additionally contain isotonic agents such as sugar or sodium chloride.

The total effective amount of the antibody or antigen-binding fragment thereof of the present invention may be administered to an individual as a single dose, or may be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. In addition, the content of the active ingredient may be varied depending on the purpose of administration. The effective dose is determined for each individual by considering various factors such as the type and severity of the target disease, the route and number of administrations, as well as the age, weight, health status, sex, disease severity, diet and excretion rate of the individual requiring administration. Therefore, a person having ordinary skill in the relevant field will be able to determine an appropriate effective dose depending on the purpose of administration. In addition, the efficacy of the therapy may be determined by monitoring the efficacy using an assay that determines the activity of immune cells after administering the antibody or fragment thereof according to the present invention or a well-known in vivo assay. The pharmaceutical composition of the present invention is not particularly limited in its formulation, route of administration and method of administration as long as it exhibits the effects of the present invention.

In addition, the present invention provides a composition for diagnosing cancer comprising the antibody or an antigen-binding fragment thereof as an active ingredient.

The antibody or antigen-binding fragment thereof and the cancer are as described above.

The term "diagnosis," as used herein, refers to the assessment of the presence or nature of a pathological condition. For the purposes of the present invention, diagnosis refers to determining the presence of cancer.

Since the antibody or antigen-binding fragment thereof specifically binds to the CCL7 protein, it can be used to detect CCL7 protein activation and/or overexpression. Accordingly, the antibody or antigen-binding fragment thereof of the present invention can be included in a diagnostic composition for diseases (e.g., cancer) associated with CCL7 activation and/or overproduction.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited to the following examples.

<Example 1> Selection of CCL7 human antibodies

Two types of panning were performed using synthetic human antibody phage display libraries (KFab-I, KFab-II) (Fig. 1, Fig. 3).

As shown in Figure 1, in the first panning, the CCL7 protein-immobilized immunoassay tube was blocked with 5% skim milk, and then the phage library and histone were added for reaction, and then washed with PBST (0.05% Tween 20 in PBS) to remove unbound phages. The washed immunoassay tube was eluted with 0.2 M Glycine-HCl (pH 2.2) and neutralized with 1 M Tris-HCl (pH 9.0). The neutralized phages were infected into TG1 Escherichia coli cells (OD ₆₀₀ = 0.6) and plated on solid medium containing 2% glucose and 100 μg/mL ampicillin, followed by incubation at 37°C for 16 h. The infected cells were harvested from the medium and used for phage amplification, and the amplified phages were used as input phages for the next round. This process was repeated four times in total. As shown in Fig. 3, in the second panning, 3% BSA (bovine serum albumin) was used as a blocking buffer and the histone used in the first panning method to reduce nonspecific binding was omitted, and the remaining steps were performed in the same manner as the first panning method. In each round, a PBS control panning without immobilized CCL7 protein was performed simultaneously, and the output titer was calculated, and the results are shown in Fig. 2 (first panning) and Fig. 4 (second panning). In the first panning, the output titer increased up to 23-fold compared to the control in the fourth round, confirming that phage clones for CCL7 were enriched (Fig. 2). In the second panning, the output titer increased 2.2-fold compared to the control in the fourth round, confirming that phage clones for CCL7 were enriched (Fig. 4).

Next, phage ELISA screening was performed to select monoclonal phages that bind to CCL7. A 96-well ELISA plate containing 30 ng of CCL7 protein per well was blocked with 5% skim milk or 3% bovine serum albumin (BSA). The monoclonal phages obtained through panning were added for reaction and washed with PBST (0.05% Tween 20 in PBS) to remove unbound phages. The washed plate was reacted with anti-M13-HRP antibody diluted 1:5000 and washed with PBST. The washed plate was added with TMB (3,3',5,5'-tetramethyl-benzidine) substrate solution for reaction and terminated after 8 minutes by adding 17% phosphoric acid. Absorbance was measured at 450 nm using a microplate reader, and as shown in Figures 5 and 6, the gene sequences of clones showing absorbances of 0.5 or higher were analyzed, and two Fab antibodies (1D12, 1C1) that bind to CCL7 were selected.

The amino acid or base sequence information for the heavy chain variable region and light chain variable region of each antibody according to the sequence analysis results is shown in Tables 1 to 4 below, and the underlined portions in the tables indicate complementarity determining regions (CDRs).

1D12 Sequence information Sequence number light chain variable region CDR1 RASQDISNWLN 1 light chain variable region CDR2 GASRLQS 2 light chain variable region CDR3 QQAYSFPLT 3 Heavy chain variable region CDR1 SFAMH 4 Heavy chain variable region CDR2 AIGSGGSTYYADSVKG 5 Heavy chain variable region CDR3 ETRWEFDF 6 light chain variable region
DIQMTQSPSSLSASVGDRVTITC RASQDISNWLN WYQQKPGKAPKLLIY GASRLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQAYSFPLT FGQGTKVEIK 13 heavy chain variable region
EVQLVESGGGLVQPGGSLRLSCAASGFTFS SFAMH WVRQAPGKGLEWVS AIGSGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK ETRWEFDF WGQGTLVTVSS 14

1D12 Sequence information Sequence number light chain variable region CDR1 CGCGCTAGCCAGGATATCTCTAATTGGCTGAAC 7 light chain variable region CDR2 GGTGCATCCCGTCTGCAGTCT 8 light chain variable region CDR3 CAGCAAGCATACTCTTTTCCGCTGACG 9 Heavy chain variable region CDR1 TCTTTTGCAATGCAC 10 Heavy chain variable region CDR2 GCAATCGGTTCTGGTGGTTCTACTTACTATGCCGATTCAGTGAAGGGT 11 Heavy chain variable region CDR3 GAAACTCGTTGGGAATTCGATTTC 12 light chain variable region
GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTGGGCGATCGTGTGACAATTACTTGT CGCGCTAGCCAGGATATCTCTAATTGGCTGAAC TGGTATCAGCAGAAACCGGGCAAGGCGCCAAAATTGCTGATTTAC GGTGCATCCCGTCTGCAGTCT GGTGTACCGTCCCGTTTCTCTGGCAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGCCTCCAGCCTGAAGATTTTGCCACCTATTATTGT CAGCAAGCATACTCTTTTCCGCTGACG TTCGGGCAGGGAACTAAAGTGGAAATTAAA 15 heavy chain variable region
GAAGTACAGTTGGTCGAAAGTGGCGGTGGCCTCGTGCAACCGGGTGGTTCACTGCGTCTGAGCTGCGCCGCCTCGGGTTTTACTTTCTCT TCTTTTGCAATGCAC TGGGTTCGTCAGGCGCCGGGCAAGGGTCTCGAATGGGTTTCA GCAATCGGTTCTGGTGGTTCTACTTACTATGCCGATTCAGTGAAGGGT CGCTTTACCATTTCCCGTGACAACTCTAAGAATACTCTGTATCTGCAGATGAACTCGCTGCGTGCCGAAGACACGGCCGTCTATTATTGCGCCAAA GAAACTCGTTGGGAATTCGATTTC TGGGGTCAGGGCACTTTAGTGACGTCTCATCG 16

1C1 Sequence information Sequence number light chain variable region CDR1 RASQSISNYLN 17 light chain variable region CDR2 AASRLQS 18 light chain variable region CDR3 QQSYSFPWT 19 Heavy chain variable region CDR1 EYYIH 20 Heavy chain variable region CDR2 WITPYSGVTSYAQKFQG 21 Heavy chain variable region CDR3 HEVYWYIGAFDY 22 light chain variable region
DIQMTQSPSSLSASVGDRVTITC RASQSISNYLN WYQQKPGKAPKLLIY AASRLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC QQSYSFPWT FGQGTKVEIK 29 heavy chain variable region
QVQLVQSGAEVKKPGSSVKVSCKASGGTFS EYYIH WVRQAPGQGLEWMG WITPYSGVTSYAQKFQG RVTITADESTSTAYMELSSLRSEDTAVYYCAR HEVYWYIGAFDY WGQGTLVTVSS 30

1C1 Sequence information Sequence number light chain variable region CDR1 CGCGCTAGCCAGTCTATCTCTAATTACCTGAAC 23 light chain variable region CDR2 GCAGCATCCCGTCTGCAGTCT 24 light chain variable region CDR3 CAGCAATCTTACTCTTTTCCGTGGACG 25 Heavy chain variable region CDR1 GAATACTACATCCAT 26 Heavy chain variable region CDR2 TGGATTACCCCATACTCTGGTGTTACCTCTTATGCACAAAAATTCCAAGGC 27 Heavy chain variable region CDR3 CATGAAGTTTACTGGTACATCGGTGCATTCGATTAC 28 light chain variable region
GACATTCAAATGACGCAGAGTCCCTCCTCACTGAGTGCTAGCGTGGGCGATCGTGTGACAATTACTTGT CGCGCTAGCCAGTCTATCTCTAATTACCTGAAC TGGTATCAGCAGAAACCTGGCAAGGCGCCAAAATTGCTGATTTAC GCAGCATCCCGTCTGCAGTCT GGTGTACCGTCCCGTTTCTCTGGCAGCGGTTCTGGTACGGATTTTACCCTGACCATCTCAAGCCTCCAGCCTGAAGATTTTGCCACCTATTATTGT CAGCAATCTTACTCTTTTCCGTGGACG TTCGGGCAGGGAACTAAAGTGGAAATTAAA 31 heavy chain variable region
CAAGTTCAGCTGGTCCAGAGCGGCGCAGAGGTGAAGAAGCCCGGCAGTTCTGTTAAGGTTTCCTGCAAAGCCTCAGGCGGGACTTTTAGT GAATACTACATCCAT TGGTGCGGCAGGCGCCCGGCCAGGGTCTCGAATGGATGGGG TGGATTACCCCATACTCTGGTGTTACCTCTTATGCACAAAAATTCCAAGGC CGCGTAACTATTACCGCCGACGAATCAACCTCCACCGCCTACATGGAACTCAGCTCTCTGAGGTCAGAAGACACGGCCGTCTATTATTGCGCCAGA CATGAAGTTTACTGGTACATCGGTGCATTCGATTAC TGGGGTCAGGGTACTCTGGTTACCGTCTCATCG 32

<Example 2> Production of CCL7 human antibody

Experiments were performed to convert two selected Fab antibodies (1D12, 1C1) into the human IgG1 form and to verify this.

Specifically, the selected YKL-40 phage was transfected into Expi293 cells, cultured, and IgG antibody protein was purified from 300 ml of the cultured Expi293 cell culture fluid. After purification using a HiTrap MabSelect SuRe column, the purified IgG was analyzed for molecular weight and purity by SDS-PAGE.

As a result, as shown in Fig. 7, a band of 25 kDa for the light chain and 50 kDa for the heavy chain was confirmed under reducing conditions, and a band of 150 kDa in which two light chains and two heavy chains were linked by disulfide bonds was confirmed under non-reducing conditions. In other words, the molecular weights of the light and heavy chains of the 1D12 and 1C1 antibodies were confirmed to be consistent with the theoretical calculations.

In addition, as shown in FIGS. 8 and 9, it was confirmed through SE-HPLC (Size exclusion-high-performance liquid chromatography) analysis that the monomer peak ratio was 93% or more and no aggregation peak was found.

<Example 3> Confirmation of antigen binding ability of CCL7 human antibody

An experiment was conducted to analyze the antigen binding ability of the two types of antibodies produced (1D12 IgG1, 1C1 IgG1).

Specifically, a 96-well ELISA plate with 60 ng of CCL7 protein immobilized per well was blocked with 3% BSA (bovine serum albumin), and then two types of antibodies (1D12 IgG1, 1C1 IgG1) diluted at different concentrations were added for reaction, and unbound antibodies were removed by washing with PBST (0.05% Tween 20 in PBS). Anti-human IgG Fc-HRP antibody diluted 1:10000 was added to the washed plate, reaction was performed, and washing with PBST. TMB (3,3',5,5'-tetramethyl-benzidine) substrate solution was added to the washed plate, reaction was performed, and 17% phosphoric acid was added after 4 minutes to terminate the reaction. The absorbance was measured at 450 nm using a microplate reader, and the obtained absorbance data were plotted using 4-parameter logistic regression analysis to calculate the EC ₅₀ value. At this time, the antigen binding capacity of the antibody was calculated as the amount required to reach 50% of the maximum binding (EC ₅₀ ).

As a result, as shown in Fig. 10, the EC ₅₀ values were 124.1 pM for the 1D12 antibody and 77.3 pM for the 1C1 antibody, confirming that both antibodies exhibited excellent binding ability to CCL7.

<Example 4> Confirmation of binding affinity of CCL7 human antibody to antigen

An experiment was performed to analyze the affinity of two types of antibodies (1D12 IgG1, 1C1 IgG1) for the antigen.

Specifically, the measurement was performed through SPR analysis using CCL7 recombinant protein. The analysis was performed by immobilizing the antibody on the chip, and the K _on , K _off , and K _D values were obtained using BIA evaluation software.

As a result, as shown in Figure 11, the K _D values indicating affinity for CCL7 were confirmed to be 2.8 nM for the 1D12 antibody and 3.61 nM for the 1C1 antibody. Both types of antibodies exhibited high antigen binding affinity at the nanomolar level.

<Example 5> Evaluation of the efficacy of CCL7 human antibodies: chemotaxis assay

To determine whether human CCL7 antibodies can inhibit CCL7-induced chemotaxis of human monocytes, an in vitro chemotaxis assay was performed.

Specifically, the extent to which chemotaxis induced by treating human recombinant CCL7 at 0.25 μg/ml in human monocyte cell line THP-1 cells was inhibited by a novel human CCL7-binding antibody was confirmed through a transwell system.

As a result, as shown in Figures 12 and 13, concentration-dependent chemotaxis inhibition was confirmed in the groups treated with the positive control antibody (α-hCCL7) and the human CCL7-binding novel antibody (1D12 IgG1).

<Example 6> Evaluation of the efficacy of CCL7 human antibody: cancer cell proliferation assay

<6-1> Increased cancer cell growth by CCL7

To determine the effect of CCL7 on cancer cell growth, a cancer cell proliferation assay was performed.

Specifically, human colon cancer cell line HT-29 cells were seeded at 1x10 ³ cells/well in a 96-well plate, and then treated with human recombinant CCL7 at concentrations of 0, 20, and 200 ng/ml for 48 hours. The changes in the proliferation rate of cancer cells were measured using the WST-1 assay.

As a result, as shown in Fig. 14, it was confirmed that the proliferation rate of cancer cells treated with CCL7 at a concentration of 20 ng/ml increased.

<6-2> Inhibition of cancer cell growth by novel human CCL7-binding antibodies

To determine whether the novel human CCL7-binding antibody is effective in inhibiting the growth of cancer cells induced by CCL7, a cancer cell proliferation assay was performed.

Specifically, human colon cancer cell line HT-29 cells were seeded in 96-well plates at ^1x103 cells/well, and then treated with human recombinant CCL7 at concentrations of 0 and 20 ng/ml for 48 hours. After treating the cancer cells with human recombinant CCL7 and a novel human CCL7-binding antibody to neutralize CCL7, the changes in cancer cell proliferation rate were measured. MAB282CCL7 (neutralized antibody, R&D systems) was used as a positive control.

As a result, as shown in Figures 15 and 16, it was confirmed that when only CCL7 was treated at a concentration of 20 ng/ml, the growth of cancer cells increased, but when treated together with two types of human CCL7-binding novel antibodies, the growth of cancer cells did not increase.

Attach an electronic file of the sequence list

Claims (15)

An antibody or antigen-binding fragment thereof that specifically binds to CCL7 (CC motif Chemokine ligand 7).
In the first paragraph,
An antibody or antigen-binding fragment thereof, characterized in that the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a light chain CDR1 defined by the amino acid sequence of SEQ ID NO: 1, a light chain CDR2 defined by the amino acid sequence of SEQ ID NO: 2, and a light chain CDR3 defined by the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising a heavy chain CDR1 defined by the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 defined by the amino acid sequence of SEQ ID NO: 5, and a heavy chain CDR3 defined by the amino acid sequence of SEQ ID NO: 6.
In the second paragraph,
An antibody, characterized in that the antibody or antigen-binding fragment thereof comprises a light chain variable region having an amino acid sequence of SEQ ID NO: 13 and a heavy chain variable region having an amino acid sequence of SEQ ID NO: 14.
In the first paragraph,
An antibody or antigen-binding fragment thereof, characterized in that the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising a light chain CDR1 defined by the amino acid sequence of SEQ ID NO: 7, a light chain CDR2 defined by the amino acid sequence of SEQ ID NO: 8, and a light chain CDR3 defined by the amino acid sequence of SEQ ID NO: 9; and a light chain variable region comprising a heavy chain CDR1 defined by the amino acid sequence of SEQ ID NO: 10, a heavy chain CDR2 defined by the amino acid sequence of SEQ ID NO: 11, and a heavy chain CDR3 defined by the amino acid sequence of SEQ ID NO: 12.
In paragraph 4,
An antibody or antigen-binding fragment thereof, characterized in that the antibody or antigen-binding fragment thereof comprises a light chain variable region having an amino acid sequence of SEQ ID NO: 29 and a heavy chain variable region having an amino acid sequence of SEQ ID NO: 30.
In the first paragraph,
An antibody or antigen-binding fragment thereof, characterized in that the fragment is a fragment selected from the group consisting of diabody, Fab, Fab′, F(ab)2, F(ab′)2, Fv and scFv.
A nucleic acid encoding the antibody of claim 1 or an antigen-binding fragment thereof.
In paragraph 7,
A nucleic acid characterized in that the nucleic acid comprises a light chain nucleic acid sequence comprising a light chain CDR1 nucleic acid sequence of SEQ ID NO: 7, a light chain CDR2 nucleic acid sequence of SEQ ID NO: 8, and a light chain CDR3 nucleic acid sequence of SEQ ID NO: 9, and a heavy chain nucleic acid sequence comprising a heavy chain CDR1 nucleic acid sequence of SEQ ID NO: 10, a heavy chain CDR2 nucleic acid sequence of SEQ ID NO: 11, and a heavy chain CDR3 nucleic acid sequence of SEQ ID NO: 12.
In paragraph 7,
A nucleic acid characterized in that the nucleic acid comprises a light chain nucleic acid sequence comprising a light chain CDR1 nucleic acid sequence of SEQ ID NO: 23, a light chain CDR2 nucleic acid sequence of SEQ ID NO: 24, and a light chain CDR3 nucleic acid sequence of SEQ ID NO: 25, and a heavy chain nucleic acid sequence comprising a heavy chain CDR1 nucleic acid sequence of SEQ ID NO: 26, a heavy chain CDR2 nucleic acid sequence of SEQ ID NO: 27, and a heavy chain CDR3 nucleic acid sequence of SEQ ID NO: 28.
A vector comprising the nucleic acid of claim 7.
A host cell comprising the vector of claim 10.
A step of culturing the host cell of claim 11; and
A method for producing an antibody or antigen-binding fragment thereof that specifically binds to CCL7, comprising the step of recovering the antibody or antigen-binding fragment thereof from the cultured cells.
A pharmaceutical composition for preventing or treating cancer, comprising the antibody of claim 1 or an antigen-binding fragment thereof as an active ingredient.
In Article 13,
A pharmaceutical composition characterized in that the cancer is selected from the group consisting of colon cancer, lung cancer, liver cancer, stomach cancer, esophageal cancer, pancreatic cancer, gallbladder cancer, kidney cancer, bladder cancer, prostate cancer, testicular cancer, cervical cancer, endometrial cancer, choriocarcinoma, ovarian cancer, breast cancer, thyroid cancer, brain cancer, head and neck cancer, skin cancer, rectal cancer, oral cavity, pharyngeal cancer, laryngeal cancer, colon cancer, bone cancer, brain tumor, thyroid cancer, leukemia, lymphoma, and multiple myeloid blood cancer.
A composition for diagnosing cancer, comprising the antibody of claim 1 or an antigen-binding fragment thereof as an active ingredient.