Fusion protein comprising Anti-CD300c antibody or antigen-binding fragment thereof and il-7, and use thereof

Abstract

The present invention relates to: a fusion protein comprising an anti-CD300c antibody or an antigen-binding fragment thereof, and IL-7; a polynucleotide encoding the fusion protein; a host cell comprising the fusion protein or the polynucleotide encoding the fusion protein; and a composition for preventing or treating cancer, the composition comprising the fusion protein.

Description

Anti-CD300C antibody or its antigen-binding fragment and a fusion protein containing IL-7 and its uses

[0001] The present invention relates to a fusion protein comprising an anti-CD300c antibody or an antigen-binding fragment thereof and IL-7, a polynucleotide encoding said fusion protein, a host cell comprising said fusion protein or the polynucleotide encoding said fusion protein, a pharmaceutical composition for cancer prevention or treatment comprising said fusion protein, and a food composition for cancer prevention or improvement comprising said fusion protein.

[0002]

[0003] Cancer is one of the diseases accounting for the largest proportion of deaths among modern people. It is a disease caused by the transformation of normal cells due to genetic mutations resulting from various causes, and it can be a malignant tumor that does not follow the differentiation, proliferation, and growth patterns of normal cells. Cancer is characterized by "uncontrolled cell growth," and this abnormal growth leads to the formation of a mass of cells called a tumor, which infiltrates surrounding tissues and, in severe cases, metastasizes to other organs of the body. Even with treatment such as surgery, radiation, and drug therapy, cancer often fails to achieve a fundamental cure, causing suffering to patients and ultimately leading to death; it is an intractable chronic disease.

[0004] Although targeted anticancer drugs and immune checkpoint inhibitors have been developed to treat such cancers, there are limitations such as the occurrence of drug resistance or low efficacy in treating cancer, so it is necessary to develop more effective anticancer drugs to overcome the aforementioned limitations (Republic of Korea Published Patent No. 10-2018-0099557).

[0005] Against this background, the inventors completed the present invention by confirming that an anti-CD300c antibody or its antigen-binding fragment and a fusion protein containing IL-7 have a synergistic effect for cancer treatment.

[0006]

[0007] The problem to be solved by the present invention is to provide a fusion protein comprising an anti-CD300c antibody or an antigen-binding fragment thereof; and IL-7.

[0008] One object of the present invention is to provide a fusion protein comprising an anti-CD300c antibody or an antigen-binding fragment thereof; and IL-7.

[0009] Another objective of the present invention is to provide a polynucleotide encoding the fusion protein.

[0010] Another objective of the present invention is to provide a vector comprising a polynucleotide encoding the fusion protein.

[0011] Another object of the present invention is to provide a host cell comprising one or more of the fusion protein; a polynucleotide encoding the same; and a vector comprising the polynucleotide.

[0012] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cancer comprising the fusion protein.

[0013] Another objective of the present invention is to provide a food composition for preventing or improving cancer, comprising the fusion protein.

[0014] Another objective of the present invention is to provide a method for preventing or treating cancer, comprising the step of administering the fusion protein to an individual.

[0015] Another object of the present invention is to provide a method for producing a fusion protein comprising the step of culturing a host cell in a medium comprising one or more of the fusion protein; a polynucleotide encoding the same; and a vector comprising the polynucleotide.

[0016] The fusion protein comprising the anti-CD300c antibody or its antigen-binding fragment and IL-7 of the present invention can exert a synergistic effect for cancer treatment by inducing anticancer immune activation through the regulation of the CD300c response, while simultaneously enhancing T-cell-based anticancer immune responses by restoring IL-7 levels reduced by treatment with the anti-CD300c antibody. Through this, the fusion protein of the present invention can effectively prevent, improve, or treat cancer.

[0017] Figure 1 is a figure confirming the binding affinity of an anti-CD300c monoclonal antibody to a CD300c antigen by ELISA.

[0018] Figure 2 is a figure confirming the binding affinity of an anti-CD300c monoclonal antibody to a CD300c antigen by surface plasmon resonance (SPR).

[0019] Figure 3 is a figure showing the binding affinity of the anti-CD300c monoclonal antibody to 293-T cells under exogenous conditions, confirmed by FACS (fluorescence-activated cell sorting).

[0020] Figure 4 is a figure confirming the binding specificity of the anti-CD300c monoclonal antibody against the CD300c antigen and family proteins.

[0021] Figure 5 shows the results of flow cytometry analysis of various macrophage markers in THP-1 cells using an anti-CD300c monoclonal antibody.

[0022] Figures 6 and 7 show the secretion of M1 macrophage-associated inflammatory cytokines by the anti-CD300c monoclonal antibody.

[0023] Figure 8 shows the results of a flow cytometry (FACS) histogram of changes in PD-1 and PD-L1 expression in THP-1 cells.

[0024] Figure 9 is a figure showing the morphological changes in human M1 macrophages treated with an anti-CD300c monoclonal antibody, observed under an optical microscope (200x magnification).

[0025] Figure 10 shows the results of quantitatively evaluating primary macrophages in the anti-CD300c monoclonal antibody treatment group, LPS treatment group, isotype control group (IgG treatment group), and untreated control group.

[0026] Figure 11 shows the results of flow cytometry analysis of various macrophage markers in primary macrophages with an anti-CD300c monoclonal antibody.

[0027] Figure 12 shows the secretion amounts of TNF-α, IL-1β, and IL-8 in the anti-CD300c monoclonal antibody treatment group.

[0028] Figure 13 shows the effect of anti-CD300c monoclonal antibody on activating the MAPK (mitogen-activated protein kinase) and NF-κB (nuclear factor κB) signaling pathways.

[0029] Figure 14 is a figure evaluating tumor growth in the anti-CD300c monoclonal antibody and anti-PD-1 treatment groups.

[0030] Figure 15 shows the results of confirming the expression of M1 macrophages within tumors in mice administered with an anti-CD300c monoclonal antibody or an anti-PD-1 monoclonal antibody using an LSM 880 microscope (Carl Zeiss, Dublin, CA).

[0031] Figure 16 shows the results confirming the induction of differentiation into M1 type macrophages by the anti-CD300c monoclonal antibody and the control group.

[0032] Figure 17 shows the experimental design and results regarding the tumor growth inhibitory effect of anti-CD300c-antibody in a Lewis-lung carcinoma (LLC) model.

[0033] Figure 18 shows the results of tumor volume confirmation and survival time measurement in a Lewis-lung carcinoma (LLC) model administered with anti-CD300c antibodies.

[0034] Figure 19 shows the results of examining the expression of PCNA, a proliferation marker, in non-small cell lung cancer (NSCLC) tumor tissue upon administration of the anti-CD300C monoclonal antibody using immunohistochemical staining.

[0035] FIG. 20 is a figure showing the experimental protocol of Example 3.4 according to the present invention, and FIG. 21 and FIG. 22 are figures showing the results of flow cytometry analysis of lung tissue from mice carrying non-small cell lung cancer (NSCLC) according to Example 3.4.

[0036] FIG. 23 is a figure showing the experimental protocols of Examples 3.5 to 3.6 according to the present invention, and FIG. 24 is a figure showing the results of flow cytometry analysis of lung tissue from mice carrying non-small cell lung cancer (NSCLC) according to Example 3.5.

[0037] FIGS. 25 and 26 are figures showing the results of qRT-PCR for analyzing the expression of various genes related to immune regulation in the tumor microenvironment according to Example 3.6 above.

[0038] Figure 27 shows the results of confirming the anticancer effect of an anti-CD300c monoclonal antibody in a TNBC (triple-negative breast cancer) mouse model.

[0039] Figures 28 and 29 show the correlation between IL-7 and CD300c, cytokines associated with T cell activation in various cancers.

[0040] FIG. 30 is a schematic diagram of a fusion protein of the present invention according to one embodiment.

[0041] FIG. 31 is a schematic diagram of a fusion protein expression vector of the present invention according to one embodiment.

[0042] Figure 32 is a figure showing the purification results of the fusion protein of the present invention.

[0043] Figure 33 is a figure showing the results of confirming the binding affinity of the fusion protein of the present invention to CD300c.

[0044] Figure 34 is a figure showing the results of confirming the binding ability of the fusion protein of the present invention to IL-7Rα.

[0045] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions provided below. Additionally, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated by reference into this specification in their entirety to more clearly explain the level of the art to which the present invention pertains and the content of the present invention.

[0046]

[0047] One aspect of the present invention provides a fusion protein comprising an anti-CD300c antibody or an antigen-binding fragment thereof; and IL-7.

[0048]

[0049] In the present invention, the term "CD300c" refers to a type of CD300 family of cell surface cells involved in immune regulation, which may be expressed in antigen-presenting cells, cancer cells, or immune cells, but is not limited thereto.

[0050] In the present invention, the term "antibody" refers to a proteinaceous molecule capable of specifically recognizing an antigenic site, comprising an immunoglobulin or a portion thereof that is immunologically reactive with a specific antigen. The antibodies of the present invention include polyclonal antibodies, monoclonal antibodies, whole antibodies, and antibody fragments. Furthermore, the antibodies of the present invention include antibodies from mice, humans, rabbits, and rats, without being limited to their origin. The antibodies of the present invention include chimeric antibodies (e.g., humanized murine antibodies), humanized antibodies, and bivalent or bispecific molecules (e.g., bispecific antibodies), diabodies, triabodidies, tetrabodies, and minibodies. The antibodies of the present invention further include short-chain antibodies, scaffolds, derivatives of antibody constant regions, and artificial antibodies based on protein scaffolds that possess binding function to FcRn (neonatal Fc receptor). The total antibody has a structure having two total length light chains (LC) and two total length heavy chains (HC), and each light chain may be connected to the heavy chain by a disulfide bond. The total antibody includes IgA, IgD, IgE, IgM, and IgG, and IgG includes subtypes IgG1, IgG2, IgG3, and IgG4. Such antibodies may be prepared by cloning each gene into an expression vector according to a conventional method to obtain a protein encoded by said gene, and from the obtained protein by a conventional method, but are not limited thereto.

[0051] In the present invention, the term “fragment” or “antibody fragment” refers to any part of an antibody, and may include scFv, dsFv, Fab, Fab’, F(ab’)2, Fc, Fd, sdAb, nanobody, and combinations thereof, and may include an antibody fragment, and may include an antigen recognition site, but is not limited thereto.

[0052] In the present invention, the term "antigen-binding fragment" refers to a fragment possessing an antigen-binding function. In the present invention, the antigen-binding fragment may be a fragment comprising a site capable of recognizing an antigenic site.

[0053] In the present invention, Fd refers to the heavy chain portion included in the Fab fragment. In the present invention, Fab has a structure having a variable region of the light and heavy chains, a constant region of the light chain, and a first constant region of the heavy chain (CH1 domain), 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. The F(ab')2 antibody is generated when the cysteine ​​residues in the hinge region of Fab' form disulfide bonds. Fv (variable fragment) refers to the smallest antibody fragment having only the heavy chain variable region and the light chain variable region. In a double disulfide Fv (dsFv), the heavy chain variable region and the light chain variable region are connected by disulfide bonds, and in a single chain variable fragment (scFv), the heavy chain variable region and the light chain variable region are generally connected by covalent bonds through a peptide linker. A double disulfide single chain Fv may be a single chain Fv in which the heavy chain variable region and the light chain variable region are additionally connected by disulfide bonds. The above sdAb and nanobody are single variable domain antibody fragments, and may include, for example, antibody fragments produced by protein hydrolysis or genetic recombination technology among heavy chain antibodies containing a naturally occurring single variable domain (VH) and two constant domains (CH2 and CH3), and single domain antibody fragments produced by artificially modifying the antibody light chain or heavy chain variable domain, but are not limited thereto. These antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restricting the whole antibody with papain and F(ab')2 fragment can be obtained by cleaving it with pepsin), or produced through genetic recombination technology.

[0054] The anti-CD300c antibody of the present invention refers to an antibody capable of binding to CD300c.

[0055] The anti-CD300c antibody of the present invention refers to an antibody capable of binding to CD300c and may regulate the reaction of CD300c.

[0056] For example, the anti-CD300c antibody of the present invention may act as an agonist of CD300c, but is not limited thereto.

[0057]

[0058] In one embodiment, the anti-CD300c antibody of the present invention may be a monoclonal antibody.

[0059] In the present invention, the term “monoclonal antibody” refers to an antibody molecule of a single molecular composition obtained from substantially the same group of antibodies, and such monoclonal antibodies exhibit single binding specificity and affinity for a specific epitope.

[0060] Typically, immunoglobulins have heavy chains and light chains, and each heavy chain and light chain includes an invariant region and a variable region. The variable regions of the light chain and heavy chain include three variable regions called complementarity-determining regions (CDRs) and four structural regions (FRs).

[0061] The above CDRs primarily serve to bind to the epitope of the antigen. The CDRs of each chain are typically named sequentially starting from the C-terminus as CDR1, CDR2, and CDR3, and are also identified by the chain in which a specific CDR is located. The complementarity determining region is situated between regions called the relatively conservative invariant region (FR). Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino-terminus to the carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with the antigen. The CDRs of the heavy chain variable region may be referred to as HCDR1, HCDR2, and HCDR3, the CDRs of the light chain variable region as LCDR1, LCDR2, and LCDR3, the FRs of the heavy chain variable region as HFR1, HFR2, HFR3, and HFR4, and the FRs of the light chain variable region as LFR1, LFR2, LFR3, and LFR4.

[0062] In one embodiment, the anti-CD300c antibody of the present invention or the antigen-binding fragment thereof may comprise, but is not limited to, the heavy chain CDR1 of SEQ ID NO. 3; the heavy chain CDR2 of SEQ ID NO. 5; the heavy chain CDR3 of SEQ ID NO. 7; the light chain CDR4 of SEQ ID NO. 11; the light chain CDR5 of SEQ ID NO. 13; and the light chain CDR6 of SEQ ID NO. 15.

[0063] In one embodiment, the heavy chain CDR1 may be coded by the polynucleotide sequence of SEQ ID NO. 4; the heavy chain CDR2 may be coded by the polynucleotide sequence of SEQ ID NO. 6; the heavy chain CDR3 may be coded by the polynucleotide sequence of SEQ ID NO. 8; the light chain CDR4 may be coded by the polynucleotide sequence of SEQ ID NO. 12; the light chain CDR5 may be coded by the polynucleotide sequence of SEQ ID NO. 14; and / or the light chain CDR6 may be coded by the polynucleotide sequence of SEQ ID NO. 16, but is not limited thereto.

[0064] In one embodiment, the anti-CD300c antibody of the present invention or the antigen-binding fragment thereof may comprise, but is not limited to, the heavy chain FR1 of SEQ ID NO. 17; the heavy chain FR2 of SEQ ID NO. 19; the heavy chain FR3 of SEQ ID NO. 21; the heavy chain FR4 of SEQ ID NO. 23; the light chain FR5 of SEQ ID NO. 25; the light chain FR6 of SEQ ID NO. 27; the light chain FR7 of SEQ ID NO. 29; and the light chain FR8 of SEQ ID NO. 31.

[0065] In one embodiment, the heavy chain FR1 may be coded by the polynucleotide sequence of SEQ ID NO. 18; the heavy chain FR2 may be coded by the polynucleotide sequence of SEQ ID NO. 20; the heavy chain FR3 may be coded by the polynucleotide sequence of SEQ ID NO. 22; the heavy chain FR4 may be coded by the polynucleotide sequence of SEQ ID NO. 24; the light chain FR5 may be coded by the polynucleotide sequence of SEQ ID NO. 26; the light chain FR6 may be coded by the polynucleotide sequence of SEQ ID NO. 28; the light chain FR7 may be coded by the polynucleotide sequence of SEQ ID NO. 30; and / or the light chain FR8 may be coded by the polynucleotide sequence of SEQ ID NO. 32, but is not limited thereto.

[0066] In one embodiment, the anti-CD300c antibody of the present invention or the antigen-binding fragment thereof may include the heavy chain variable region of SEQ ID NO. 1 and the light chain variable region of SEQ ID NO. 9, but is not limited thereto.

[0067] In one embodiment, the heavy chain variable region may be coded by the polynucleotide of SEQ ID NO. 2 and / or the light chain variable region may be coded by the polynucleotide of SEQ ID NO. 10, but is not limited thereto.

[0068] In this specification, the anti-CD300c antibody of the present invention or its antigen-binding fragment may also be referred to interchangeably as 'CL7'.

[0069] In the present invention, the variable region CDR was determined by a conventional method according to a system devised by Kabat et al. (see reference [Kabat et al., Sequences of Proteins of Immunological Interest (5th), National Institutes of Health, Bethesda, MD. (1991)]). Although the CDR numbering used in the present invention utilized the Kabat method, antibodies containing CDRs determined by other methods, such as the IMGT method, the Chothia method, and the AbM method, are also included within the scope of the present invention.

[0070] When the antibody of the present invention includes an invariant region, it may include an invariant region derived from IgG, IgA, IgD, IgE, IgM, or a combination thereof or a hybrid thereof.

[0071] In the present invention, the term “combination” means that when forming a dimer or a multimer, a polypeptide encoding a single-chain immunoglobulin constant region of the same origin forms a combination with a single-chain polypeptide of a different origin. For example, a dimer or a multimer can be formed from two or more constant regions selected from the group consisting of the constant regions of IgG, IgA, IgD, IgE, and IgM.

[0072] In the present invention, the term "hybrid" means that there are sequences corresponding to two or more immunoglobulin heavy chain constant regions of different origin within a short-chain immunoglobulin heavy chain constant region, and, for example, a hybrid of a domain consisting of one to four domains selected from the group consisting of CH1, CH2, CH3, and CH4 of IgG, IgA, IgD, IgE, and IgM is possible.

[0073] In addition, in the present invention, the origin of the variable region and the constant region of the antibody may be the same or different, and the origin of the variable region and the constant region excluding the CDR may be the same or different.

[0074]

[0075] The anti-CD300c antibody or its antigen-binding fragment of the present invention binds to CD300c receptors on the surface of immune cells and regulates the signal transduction of the corresponding receptors, thereby inducing immune cell activation and exerting a regulatory effect to exhibit an anticancer effect.

[0076] In one embodiment of the present invention, changes in the immune environment were confirmed in which the expression levels of one or more genes selected from the factors listed in Tables 4 to 36 were regulated by treatment with an anti-CD300C monoclonal antibody, and through these results, it was confirmed that the CD300c antibody affects the growth of cancer tissue.

[0077]

[0078] In the present invention, the term "IL-7 (Interleukin-7)" refers to one of the interleukins, which are a group of cytokines, and is a hematopoietic growth factor secreted from the bone marrow and the like. IL-7 is known as a glycoprotein that plays an important role in T cell activation, which primarily maintains the survival, differentiation, and proliferation of T cells. IL-7 may also be referred to as interleukin-7, interleukin-7, IL7, etc.

[0079] In one embodiment, the IL-7 of the present invention may comprise the amino acid sequence of SEQ ID NO. 33 or 35.

[0080] In one embodiment, the IL-7 of the present invention may be encoded by the polynucleotide sequence of SEQ ID NO. 34 or 36, but is not limited thereto.

[0081] The inventors have confirmed that the expression correlation between CD300c and IL-7 is significantly high in many types of cancer, and have also confirmed that in some types of cancer, the expression of IL-7 tends to decrease when a CD300c antibody is administered alone. Based on these findings, one of the main technical features of the present invention is to provide a protein fused with IL-7 and an anti-CD300c antibody or its antigen-binding fragment to achieve a synergistic effect that enhances the anticancer effect.

[0082]

[0083] The fusion protein of the present invention comprises an anti-CD300c antibody or an antigen-binding fragment thereof; and IL-7, and may be an artificially synthesized protein to which the anti-CD300c antibody or an antigen-binding fragment thereof; and IL-7 are bound, but is not limited thereto.

[0084] In this specification, the fusion protein of the present invention may also be referred to as the fusion protein 'CB701'.

[0085] In one embodiment, the fusion protein of the present invention may exhibit a synergistic effect by including both an anti-CD300c antibody or its antigen-binding fragment; and IL-7, thereby enhancing the cancer treatment effect.

[0086]

[0087] The fusion protein of the present invention may include, but is not limited to, an anti-CD300c antibody or its antigen-binding fragment; and IL-7 directly linked, linked via a linker, or additionally include other protein moiety. The linking method and linking location of the fusion protein of the present invention may utilize any method and location practiced in the art without limitation, provided that such modification does not alter the structure or activity of the linked protein.

[0088] The above direct connection may be a covalent connection, but is not limited thereto.

[0089] The above linker may be a peptide linker or a non-peptide linker, but is not limited thereto.

[0090] The above peptide linker may include one or more amino acids, for example, from 1 to 1000, specifically from 1 to 100, more specifically from 1 to 50 amino acids, but is not particularly limited thereto. Any peptide linker known in the art may include, for example, [GS]x linker, [GGGS]x linker, and [GGGGS]x linker, etc., where x may be a natural number greater than or equal to 1 (for example, 1, 2, 3, 4, 5, or more).

[0091] The above-mentioned nonpeptide linker is not limited to any type as long as it can link IL-7 with the anti-CD300c antibody or its antigen-binding fragment. For example, the above-mentioned nonpeptide linker may be selected from lipid polymers, biodegradable polymers, chitins, and oligonucleotides, but is not limited thereto.

[0092] The fusion protein of the present invention may be prepared by methods such as conjugating an anti-CD300c antibody, its antigen-binding fragment, IL-7, or a combination thereof with a substance capable of increasing the half-life, or by introducing a mutation to prevent degradation in the body, and any method known in the art that can act on the protein and increase persistence is included in the scope of the present invention. The substance capable of increasing the half-life may be selected from the group consisting of, but is not limited to, polymers, fatty acids, cholesterol, albumin and its fragments, albumin conjugates, antibodies, antibody fragments, FcRn conjugates, in vivo connective tissue, nucleotides, fibronectin, transferrin, saccharides, heparin, and elastin.

[0093] In one embodiment, the fusion protein of the present invention may include the amino acid sequence of SEQ ID NO. 37, 39, or 40, but is not limited thereto.

[0094]

[0095] The fusion protein of the present invention may simultaneously perform anticancer immune activation through the regulation of CD300c response and the enhancement of T cell activity.

[0096] The fusion protein of the present invention may maintain binding ability to CD300c equivalent to that of an anti-CD300c monoclonal antibody while not exhibiting binding ability to CD300a. In one embodiment of the present invention, it was confirmed that the fusion protein of the present invention induces sustained signal transduction and M1 macrophage differentiation by binding to the CD300c antigen, and it was also confirmed that it exhibits strong binding affinity to CD300c but does not exhibit binding ability to CD300a.

[0097] The fusion protein of the present invention may exhibit excellent binding ability to IL-7Rα, an IL-7 receptor.

[0098] The fusion protein of the present invention may improve the side effects caused by treatment with the anti-CD300c antibody alone.

[0099] The fusion protein of the present invention may enhance T-cell-based anticancer immune responses by restoring IL-7 levels that are reduced by treatment with anti-CD300c antibodies. In one embodiment of the present invention, it was confirmed that the fusion protein of the present invention can bind to CD300c to promote the differentiation of monocytes into macrophages, thereby increasing immune activity, while simultaneously binding to IL-7Rα, a receptor for IL-7, to induce T-cell activation.

[0100]

[0101] The technical feature of the fusion protein of the present invention is that it can exhibit synergy in anticancer effects through the simultaneous delivery of an anti-CD300c antibody or its antigen-binding fragment and IL-7.

[0102] The fusion protein of the present invention may have one or more of increased T cell activation ability, macrophage differentiation ability, and immune cell migration ability compared to the anti-CD300c monoclonal antibody.

[0103] The fusion protein of the present invention may have an increased cancer cell growth inhibitory effect compared to the anti-CD300c monoclonal antibody.

[0104]

[0105] Another aspect of the present invention provides a polynucleotide encoding a fusion protein of the present invention.

[0106] The above fusion protein is as described in other embodiments. The polynucleotide encoding the fusion protein of the present invention can be easily isolated and sequenced using conventional procedures.

[0107] In one embodiment, the polynucleotide encoding the fusion protein of the present invention may be codon-optimized, but is not limited thereto.

[0108] As an example of the above-described embodiment, the fusion protein of the present invention may be encoded by the sequence of SEQ ID NO. 38, but is not limited thereto.

[0109] However, considering codon degeneracy or codons preferred in organisms, it is obvious that polynucleotide sequences in which some sequences have been deleted, modified, substituted, conservatively substituted, or added are also included within the scope of the polynucleotides of the present invention, provided that such sequences can code for the amino acid sequence of the protein of the present invention or a polypeptide having homology or identity therewith.

[0110]

[0111] Another aspect of the present invention provides a vector comprising a polynucleotide encoding a fusion protein of the present invention.

[0112] Another aspect of the present invention provides a host cell comprising one or more of the following: a fusion protein of the present invention; a polynucleotide encoding the same; and a vector encoding the polynucleotide.

[0113] The above fusion protein and the polynucleotide encoding it are as described in other embodiments.

[0114] The expression vector comprising the polynucleotide encoding the fusion protein 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., E. coli, etc.). Specifically, it may be a vector comprising at least one selection marker that is operably linked to an appropriate promoter to enable the expression of the polynucleotide in a host cell. Examples include a form in which the polynucleotide is introduced into a phage, plasmid, cosmid, mini-chromosome, virus, or retroviral vector.

[0115] An expression vector comprising a polynucleotide encoding the fusion protein may be an expression vector comprising, respectively, a polynucleotide encoding the heavy chain, light chain, and IL-7 of the anti-CD300c antibody of the fusion protein or its antigen-binding fragment, and may be an expression vector comprising all polynucleotides encoding one or more of the heavy chain, light chain, and IL-7.

[0116] The host cell into which the expression vector provided in the present invention is introduced is not particularly limited thereto, but may be a bacterial cell such as Escherichia coli, Streptomyces, or Salmonella typhimurium into which the expression vector is introduced and transformed; a yeast cell; a fungal cell such as Pichia pastorlis; an insect cell such as Drozophylla or Spodoptera Sf9 cell; an animal cell such as CHO (Chinese hamster ovary cells), ExpiCHO cell, SP2 / 0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), Bowes melanoma cell, HT-1080, BHK (baby hamster kidney cells), HEK (human embryonic kidney cells), or PER.C6 (human retinal cell); or a plant cell.

[0117] In the present invention, the term "introduction" refers to the delivery of a vector containing a polynucleotide encoding the fusion protein into a host cell. Such introduction may be carried out by various methods known in the art, such as calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroshock, microinjection, liposome fusion, lipofectamine, and protoplast fusion. Additionally, the vector may be introduced into the host cell by gene bombardment, etc. In the present invention, introduction may be used interchangeably with transfection and transformation.

[0118]

[0119] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of cancer comprising a fusion protein of the present invention.

[0120] The above fusion protein is as described in other embodiments.

[0121] In the present invention, the term "cancer" refers to a disease characterized by autonomous overgrowth of cells in body tissues. The cancer is a malignant tumor formed by the abnormal growth of cells, which constitutes a mass of cells called a tumor.

[0122] The cancer of the present invention includes, without limitation, any type of cancer that can be prevented or treated by the fusion protein of the present invention, but may be a cancer expressing the CD300c protein on the surface of cancer cells or a cancer associated with CD300c expression.

[0123] Specifically, the cancer may be pancreatic cancer, stomach cancer, colorectal cancer, bile duct cancer, esophageal cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, tongue cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, lymphoma, and blood cancer.

[0124] More specifically, the cancer may include one or more selected from the group consisting of bladder cancer, breast cancer, head and neck cancer, kidney cancer, liver cancer, ovarian cancer, lung cancer, prostate cancer, and skin cancer.

[0125] More specifically, the cancer may include one or more selected from the group consisting of bladder urothelial carcinoma, invasive breast cancer, head and neck squamous cell carcinoma, chromatophoretic renal cell carcinoma and clear cell renal cell carcinoma, hepatocellular carcinoma, ovarian serous cystic adenocarcinoma, lung adenocarcinoma and lung squamous cell carcinoma, prostate adenocarcinoma, and cutaneous melanoma.

[0126] In one embodiment of the present invention, it was confirmed that CD300c and IL-7 exhibit a particularly high expression correlation in a specific cancer type. This correlation suggests that the two molecules are closely associated with the biological characteristics of the cancer type, and accordingly, it was found that the fusion protein according to the present invention can exhibit a synergistic effect of enhanced anticancer activity, particularly in the said cancer type.

[0127] As a more specific example of implementation, the cancer may be a cancer in which IL-7 expression is altered due to treatment or administration of an anti-CD300c antibody, and may be, for example, colorectal cancer, blood cancer, breast cancer, or lung cancer.

[0128]

[0129] In the present invention, the term "prevention" refers to any act of inhibiting or delaying the onset, proliferation, survival, metastasis, recurrence, and / or anticancer drug resistance of cancer by administering the above composition.

[0130] In the present invention, the term "treatment" refers to any act in which the proliferation, survival, metastasis, recurrence, and / or resistance to anticancer drugs of cancer is reduced, or the symptoms of cancer are improved or beneficially altered by the administration of the above composition.

[0131] The above pharmaceutical composition may further include a pharmaceutically acceptable carrier.

[0132] In the present invention, the term "pharmaceuticalally acceptable carrier" refers to a carrier or diluent that does not irritate living organisms and does not impair the biological activity and properties of the administered compound. Acceptable pharmaceutical carriers for compositions formulated as liquid solutions include saline solution, sterile water, Ringer's solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and mixtures of one or more of these components, provided that they are sterile and biocompatible. Additionally, other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into injectable formulations such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets.

[0133] The above pharmaceutical composition may be in various dosage forms for oral or parenteral administration. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, humectants, disintegrants, and surfactants. Solid dosage forms for oral administration include tablets, pills, powders, granules, and capsules, and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, or gelatin, with one or more compounds. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid dosage forms for oral administration include suspensions, liquids, emulsions, and syrups, and may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives, in addition to commonly used simple diluents like water and liquid paraffin. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspension solvents. Witepsol, macrogol, Tween 61, cocoa paste, laurin paste, glycerogelatin, etc. may be used as bases for suppositories.

[0134] The above pharmaceutical composition may have any one formulation selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquid formulations, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories.

[0135]

[0136] In one embodiment, the composition may contain a fusion protein in a pharmaceutically effective amount. In one example, the fusion protein may be included in an amount of about 0.001% to 100% by weight, or 0.001% to 99% by weight, of the total composition, but is not limited thereto.

[0137]

[0138] The composition of the present invention is administered in a pharmaceutically effective amount.

[0139] In the present invention, the term "pharmaceuticalally effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including individual type and severity, age, gender, type of cancer, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. It may also be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by considering all of the above factors, and this can be easily determined by a person skilled in the art.

[0140]

[0141] Another aspect of the present invention provides a method for preventing or treating cancer using the fusion protein of the present invention.

[0142] Another aspect of the present invention provides a use of the fusion protein of the present invention for cancer prevention or treatment.

[0143] The method for preventing or treating the above cancer may include the step of administering the fusion protein of the present invention or a composition containing the same to an individual suspected of having cancer.

[0144] The above fusion protein, cancer, prevention, and treatment are as described in other embodiments.

[0145] The above method may be a method comprising the step of administering a pharmaceutical composition further comprising the fusion protein of the present invention and a pharmaceutically acceptable carrier to an individual who has developed or is suspected of having developed cancer, wherein the pharmaceutically acceptable carrier is the same as described above.

[0146] The above-mentioned subjects include mammals such as cattle, pigs, sheep, chickens, dogs, and humans, birds, etc., and include without limitation subjects for whom cancer can be prevented or treated by administration of the above-mentioned composition of the present invention. In this case, the fusion protein or the composition containing it may be administered in a single or multiple doses in a pharmaceutically effective amount. In this case, the fusion protein or the composition containing it may be administered in the form of a liquid, powder, aerosol, capsule, enteric-coated tablet, or capsule or suppository. The routes of administration include, but are not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, endodermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration, etc.

[0147]

[0148] Another aspect of the present invention provides a food composition for preventing or improving cancer comprising a fusion protein of the present invention.

[0149] The above food composition may be a health functional food.

[0150] The above fusion protein and prevention are as described in other embodiments.

[0151] In the present invention, the term "improvement" refers to any act of using the fusion protein to reduce cancer proliferation, survival, metastasis, recurrence, and / or anticancer drug resistance in individuals suspected of or diagnosed with cancer, or to improve or benefit symptoms.

[0152] In the present invention, the term "food" includes all foods in the conventional sense, such as meat, sausage, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, and health functional foods, and is not limited thereto as long as it may contain the fusion protein of the present invention. When manufacturing the above food composition, raw materials and ingredients conventionally added in the art may be added, and the types thereof are not particularly limited. For example, as with conventional foods, various herbal extracts, food science-acceptable food additives, or natural carbohydrates may be included as additional ingredients, but are not limited thereto. The amount of the active ingredient can be appropriately determined according to the purpose of use.

[0153] In the present invention, the term "health functional food" is synonymous with "food for special health use (FosHU)," and refers to a food manufactured or processed for the purpose of health supplementation by using specific ingredients as raw materials or by methods such as extraction, concentration, purification, or mixing of specific ingredients contained in food raw materials. It refers to a food designed and processed to fully exert bio-regulatory functions on the body, such as biological defense, regulation of biological rhythms, and prevention and recovery from disease, through the aforementioned ingredients; and the composition for the health functional food can perform functions related to the prevention and recovery from disease. The health functional food of the present invention may be used interchangeably with terms known in the art, such as "functional food."

[0154]

[0155] Another aspect of the present invention provides a feed composition for cancer prevention or improvement comprising a fusion protein of the present invention.

[0156] The above feed composition may be for animals other than humans, and may be for said individuals, such as mammals including cattle, pigs, sheep, chickens, dogs, humans, etc., birds, etc., but is not limited thereto.

[0157] In addition to the fusion protein of the present invention, the above feed composition may include known carriers, stabilizers, or additives that are acceptable for pharmaceutical, food, or feed purposes. For example, binders, emulsifiers, preservatives, etc. are added to prevent quality degradation, and amino acid preparations, vitamins, enzymes, flavorings, non-protein nitrogen compounds, silicates, buffers, extractants, oligosaccharides, etc. are added to the feed to enhance utility. In addition, feed mixing agents, etc., may be additionally included, but are not limited thereto. The above feed composition may include various nutrients such as vitamins, amino acids, and minerals, antioxidants, and other additives as needed, and may be in a suitable form such as powder, granules, pellets, or suspension. The feed composition of the present invention may be supplied to monogastric animals alone or mixed with feed. The feed of the present invention is not particularly limited and any feed such as powder feed, solid feed, dry feed, wet feed, moist pellet feed, dry pellet feed, EP (Extruder Pellet) feed, raw feed, etc. may be used.

[0158]

[0159] Another aspect of the present invention provides a method for producing a fusion protein comprising the step of culturing a host cell in a medium comprising one or more of the following: the fusion protein of the present invention; a polynucleotide encoding the same; and a vector comprising said polynucleotide. In the method for producing the fusion protein, the culture process, medium, culture temperature, pH, etc., can be appropriately controlled using a suitable method known in the art.

[0160] The above fusion protein, polynucleotide, vector, and host cell are as described in other embodiments.

[0161] In one embodiment, the method for producing a fusion protein of the present invention may further include the steps of preparing a host cell of the present invention, preparing a medium for culturing said cell, recovering a fusion protein from the medium or host cell, purifying, or a combination thereof (in any order). The recovery and purification may also involve collecting the fusion protein using a suitable method known in the art (e.g., centrifugation, filtration, various chromatography, etc., or a combination thereof).

[0162] The present invention will be explained in more detail below through examples. However, the following examples are merely preferred embodiments for illustrating the present invention and are therefore not intended to limit the scope of the present invention. Meanwhile, technical matters not described in this specification can be fully understood and easily implemented by a person skilled in the art in the field of the present invention or a similar field.

[0163]

[0164] Example 1. Confirmation of the immune cell differentiation ability of anti-CD300c monoclonal antibody

[0165] Example 1.1 Confirmation of CD300c antigen recognition and binding of anti-CD300c monoclonal antibody thereto

[0166] Anti-CD300c monoclonal antibodies (CL7) were isolated from a synthetic human scFv phage library based on VH3-23 and VL1-47 with non-combined CDR diversity and performed four biopannings against surface-immobilized human CD300c antigens. Eighteen monoclonal scFvs were selected and converted to full-length immunoglobulin G1 versions. After transfection with sets of heavy and light chain vectors, each monoclonal antibody was expressed using the Expi-CHO expression system (A29133; Gibco) according to the manufacturer's instructions and purified into protein A beads.

[0167] The anti-CD300c antibody consists of three complementary determining regions (CDRs) (HCDR1, HCDR2, HCDR3) of the heavy chain and three complementary determining regions (CDRs) (LCDR1, LCDR2, LCDR3) of the light chain, and the amino acid sequences of each complementary determining region are as shown in Table 1.

[0168]

[0169] CDR1CDR2CDR3Heavy chainFTFSRYAMSWVR(Sequence No. 3)AISGSGGSTYYAD(Sequence No. 5)YCARSSQGIFDIW(Sequence No. 7)Light chainCSGNNIGTRRVHW(Sequence No. 11)SKNNRPSGVP(Sequence No. 13)YCAAWDDSLSGPVF(Sequence No. 15)

[0170] Example 1.2. Confirmation of binding affinity of anti-CD300c monoclonal antibody to CD300c antigen (ELISA)

[0171] CD300c antigen (250 μg / mL, 11832-H08H, Sino Biological) was diluted to 800 ng / well in 0.1 M sodium carbonate buffer (pH 9.0), coated in 100 μL aliquots onto 96-well microplates, and incubated overnight at 4°C. The following day, each well was washed three times with PBST and blocked with 200 μL of 5% skim milk buffer for 1 hour. Anti-CD300c monoclonal antibody was serially diluted tenfold starting from 10 μg / mL in PBS, and 100 μL of each concentration was added to the wells. The platelets were then reacted at room temperature for 1 hour to bind to the antigen. After the reaction, the platelets were washed three times with PBST to remove unconjugated antibodies.

[0172] For detection, 100 μL of HRP-conjugated α-human Fc specific IgG diluted 1:1000 was added to each well and reacted at room temperature for 1 hour. After washing three times with PBST, 100 μL of a 1:1 mixture of TMB and hydrogen peroxide was added to each well and reacted for 7–9 minutes, after which 50 μL of 1 N sulfuric acid was added to terminate the reaction. Absorbance was measured at 450 nm using a microplate reader (Varioskan LUX), and the results are shown in Figure 1.

[0173] As a result, as can be seen in Figure 1, the anti-CD300c monoclonal antibody bound to the CD300c antigen in a concentration-dependent manner, confirming that the antibody has high specificity and binding affinity for CD300c.

[0174]

[0175] Example 1.3. Confirmation of binding affinity of anti-CD300c monoclonal antibody to CD300c antigen (Surface Plasmon Resonance (SPR))

[0176] The binding affinity of the anti-CD300c monoclonal antibody was evaluated using Biacore T200 (Cytiva, Marlborough, MA). Human CD300c antigen was immobilized on the surface of a CM5 sensor chip, and the binding reaction was measured in a flow cell by injecting a running buffer at a flow rate of 10 μL / min. Ligand regeneration was achieved by denaturing the ligand with a 50 mM NaOH solution and then equilibrating it with the same running buffer. The binding rate constant (Kon) and dissociation rate constant (Koff) were calculated using Biacore T200 software (version 3.2), and the dissociation constant (KD) was calculated from the Koff / Kon value, the result of which is shown in Figure 2.

[0177] As a result, the KD value is 5.199 × 10⁻⁶ 10 It was found to be M, confirming that the binding affinity of the anti-CD300c monoclonal antibody is very high at the level of 0.52 nM. These results suggest that the anti-CD300c monoclonal antibody forms a stable and strong binding to the CD300c antigen, thereby effectively performing physiological functions such as intracellular signal transduction and the induction of M1 macrophage differentiation.

[0178]

[0179] Example 1.4. Confirmation of binding affinity of anti-CD300c monoclonal antibody to 293-T cells under exogenous conditions (FACS (fluorescence-activated cell sorting))

[0180] Human CD300c (hCD300c) binding was evaluated using 293T cells. The entire human CD300c gene was inserted into the pcDNA3.1 vector (Thermo Fisher Scientific, Waltham, MA), and CD300c was overexpressed in 293T cells by transfecting them with Lipofectamine 2000 reagent (Invitrogen). For FACS binding analysis, wild-type and hCD300c overexpressing cells were collected using dissociation buffer, and 2 × 10⁵ cells were stained as each sample. The anti-CD300c monoclonal antibody was initially diluted to 10 μg / mL in FACS buffer, and the final concentration was prepared using a 3-fold serial dilution method. Subsequently, FITC-conjugated anti-hIgG (H₅+L) was added, and the antibody was stained at room temperature for 30 minutes. After staining, the cells were washed twice with FACS buffer and analyzed using a CytoFLEX flow cytometer (Beckman Coulter). The collected data were processed using CytExpert software (Beckman Coulter), and the results are shown in Figure 3.

[0181] Consequently, as shown in Figure 3, the anti-CD300c monoclonal antibody strongly bound to CD300c overexpressed on the surface of 293T cells, while no binding was observed in wild-type cells. This suggests that the anti-CD300c monoclonal antibody possesses high specificity and binding ability to the CD300c antigen, and performs cell surface signaling and immune regulatory functions through antigen-specific binding.

[0182]

[0183] Example 1.5. Confirmation of binding specificity of anti-CD300c monoclonal antibody against CD300c antigen

[0184] Conjugation ELISA was performed to evaluate whether the anti-CD300c monoclonal antibody binds specifically only to CD300c without binding to B7 family proteins. 96-well microplates were coated with human recombinant CD300c protein, human PD-L1, PD-L2, B7-H2 (ICOS-L), B7-H3, B7-H4, B7-1 (CD80), and B7-2 (CD86) proteins at a concentration of 800 ng / mL (Sino Biological, Beijing, China) and incubated overnight at 4°C. Subsequently, blocking was performed using skim milk, after which the anti-CD300c monoclonal antibody was diluted tenfold in the range of 0.001–10 μg / mL and reacted for 1 hour. After the reaction, the sample was washed with PBST, and 100 μL of HRP-conjugated anti-human IgG antibody (Sigma-Aldrich) was added to continue the reaction for 1 hour. Subsequently, after washing with PBST, TMB (3,3′,5,5′-tetramethylbenzidine) solution was added as a substrate, and the reaction was stopped with 1 N H₂SO₄. Absorbance was measured at 450 nm using a VersaMax microplate reader, and the results are shown in Figure 4.

[0185] As a result, as can be seen in Figure 4, the anti-CD300c monoclonal antibody specifically bound only to CD300c without binding to other similar proteins. These results indicate that the anti-CD300c monoclonal antibody selectively recognizes the CD300c antigen and can induce sustained signal transduction and M1 macrophage differentiation by stably binding to the cell surface CD300c through high binding affinity.

[0186]

[0187] Example 1.6. Confirmation of Induction of Expression of Various M1 Macrophage Markers on the Surface of THP-1 Cells by Anti-CD300c Monoclonal Antibody

[0188] Next, we intended to perform flow cytometry analysis of various macrophage markers in THP-1 cells using the anti-CD300c monoclonal antibody.

[0189] To confirm the polarization of THP-1 cells, cell surface expression of the M0 marker CD11b, M1 markers CD80 and CD197 (CD197 / CCR7), and M2 marker CD206 was evaluated by flow cytometry (FACS). THP-1 cells were seeded into 96-well plates at a density of 2 × 10⁵ cells per well. Subsequently, M1 macrophage differentiation was induced by treating the cells with 100 ng / mL of LPS (Sigma-Aldrich) or 10 μg / mL of anti-CD300c monoclonal antibody for 48 hours. After treatment, cells were recovered using dissociation buffer and stained with FITC anti-human CD11b (BD Pharmingen), PE anti-human CD80 (eBioscience), PE anti-human CCR7 (CD197) (BD Pharmingen), APC anti-human CD206 (BD Pharmingen), FITC anti-human CD279 (PD-1) (BD Pharmingen), and APC anti-human CD274 (PD-L1) (BD Pharmingen) antibodies. After staining, the cells were washed with FACS buffer and analyzed using a CytoFLEX flow cytometer (Beckman Coulter), and the data were processed using the CytoExpert program (Beckman Coulter). The results are shown in Figure 5.

[0190] In the group treated with the anti-CD300c monoclonal antibody, CD11b expression increased compared to the control group, and the mean fluorescence intensity (MFI) of the M1 marker CD80 increased by approximately 6.8 times compared to the control group. The expression of an additional M1 marker, CD197, also showed an increasing trend, whereas the expression of the M2 marker, CD206, remained at a level similar to that of the control group.

[0191] These results demonstrate that the anti-CD300c monoclonal antibody polarizes THP-1 cells toward M1 macrophages, indicating that it strongly differentiates monocytes into M1 macrophages with anti-inflammatory and anticancer properties.

[0192]

[0193] Example 1.7. Confirmation of M1 Macrophage-Associated Inflammatory Cytokine Secretion by Anti-CD300c Monoclonal Antibody

[0194] To verify functional differentiation into M1 macrophages, the secretion of major inflammatory cytokines (pro-inflammatory cytokines) in the culture supernatant was measured using ELISA. The concentrations of TNF-α, IL-1β, and IL-8 in the culture supernatants of THP-1 cells and primary macrophages were analyzed using the Quantikine ELISA kit (R&D Systems, Minneapolis, MN).

[0195] First, THP-1 cells were seeded into a 96-well culture plate at a cell density of 1.5 × 10⁴. Subsequently, cells were treated with LPS to induce M1 macrophage activation, and treated with PMA (Sigma-Aldrich), IL-4 (Peprotech, Rocky Hill, NJ), and IL-13 (Peprotech) to induce M2 macrophage activation. Next, anti-CD300c monoclonal antibodies were applied to the complete culture medium, and the supernatant was collected. The concentrations of TNF-α, IL-1β, and IL-8 in the collected supernatant were measured using a Quantikine ELISA kit according to the manufacturer's instructions, and the results are shown in Figures 6 and 7.

[0196] As a result, the secretion of TNF-α, IL-1β, and IL-8 tended to increase in the group treated with the anti-CD300c monoclonal antibody, indicating that the anti-CD300c monoclonal antibody can activate macrophages into the M1 state, which possesses anti-inflammatory and anticancer properties. These results confirm that the anti-CD300c monoclonal antibody effectively contributes to the functional differentiation of macrophages and the regulation of immune responses.

[0197]

[0198] Example 1.8. Flow Cytometry (FACS) Histogram Evaluation of Changes in PD-1 and PD-L1 Expression in THP-1 Cells

[0199] The effect of anti-CD300c monoclonal antibodies on the cell surface expression of immune checkpoint proteins PD-1 and PD-L1 in human monocyte lineage cell line (THP-1) was evaluated. THP-1 cells were seeded in 96-well plates at a density of 2 × 10⁵ cells per well and M1 macrophage differentiation was induced by treatment with 100 ng / mL of LPS (Sigma-Aldrich) and 10 μg / mL of anti-CD300c monoclonal antibody for 48 hours. After treatment, cells were harvested using dissociation buffer and stained with FITC anti-human CD279 (PD-1) (BD Pharmingen) and APC anti-human CD274 (PD-L1) (BD Pharmingen) antibodies. After staining, the samples were washed with FACS buffer and analyzed using a CytoFLEX flow cytometer (Beckman Coulter). The data were processed using the CytoExpert program (Beckman Coulter), and the results are shown in Figure 8.

[0200] Flow cytometry results showed that PD-L1 expression increased in the group treated with the anti-CD300c monoclonal antibody, while no significant change was observed in PD-1 expression levels. This demonstrates that the anti-CD300c monoclonal antibody can selectively regulate the expression of some immune checkpoint markers, suggesting its potential for use in regulating immune responses within the tumor microenvironment and countering immune evasion mechanisms in cancer cells.

[0201]

[0202] Example 1.9. Morphological changes in human M1 macrophages by treatment with anti-CD300c monoclonal antibody

[0203] After 48 hours of treatment with the anti-CD300c monoclonal antibody, the morphology of primary macrophages was observed using an optical microscope (200x magnification). As a result, as shown in Figure 9, the primary macrophages of the antibody-treated group exhibited a rounded shape characteristic of human M1 macrophages, which was similar to the morphology observed in the LPS-treated group. In contrast, no such morphological change was observed in the untreated control group and the isotype control group.

[0204] These observations suggest that the anti-CD300c monoclonal antibody composition exhibits a biological activity that induces primary macrophages to adopt the M1 phenotype, thereby confirming the basis for its potential application in immunomodulation and anticancer treatment.

[0205]

[0206] Example 1.10. Confirmation of increased ratio in human M1 macrophages by treatment with anti-CD300c monoclonal antibody

[0207] Primary macrophages were treated with 100 ng / mL of LPS (Sigma-Aldrich) and 10 μg / mL of anti-CD300c monoclonal antibody for 48 hours to induce M1 macrophage differentiation. After treatment, cells were recovered using dissociation buffer, and a gating strategy was defined based on the FSC-SSC scatter plot to quantitatively evaluate primary macrophages of the anti-CD300c monoclonal antibody treatment group, LPS treatment group, isotype control group (IgG treatment group), and untreated control group via FACS analysis. The analysis results are shown in Figure 10.

[0208] Analysis results showed that morphological changes and quantitative characteristics unique to M1 macrophages were observed in the anti-CD300c monoclonal antibody-treated group, which were similar to those observed in the LPS-treated group. These changes were not observed in the untreated control group or the isotype control group.

[0209] These results indicate that the anti-CD300c monoclonal antibody has a biological activity that differentiates primary macrophages into the M1 phenotype, providing grounds for its potential application in immunomodulation and anticancer treatment.

[0210]

[0211] Example 1.11. Flow cytometry of anti-CD300c monoclonal antibody against various macrophage markers in primary macrophages

[0212] To confirm the phenotypic differentiation of primary macrophages derived from human peripheral blood monocytes (PBMCs), cell surface expression of the M0 marker CD11b, M1 markers CD80 and CD197, and M2 marker CD206 was evaluated by flow cytometry (FACS). Primary macrophages were seeded into 24-well plates at a density of 1 × 10⁶ cells per well and subsequently treated for 48 hours with 100 ng / mL of LPS (Sigma-Aldrich) and 10 μg / mL of anti-CD300c monoclonal antibody to induce M1 differentiation. After treatment, cells were collected in dissociation buffer and stained with PE anti-human CD80 (eBioscience) and PE anti-human CCR7 (CD197) (BD Pharmingen). After washing, analysis was performed using a CytoFLEX flow cytometer (Beckman Coulter), and the data were processed using the CytoExpert program (Beckman Coulter).

[0213] Analysis of the gated cell population revealed that the macrophage ratio in the anti-CD300c monoclonal antibody-treated group was 47.4%, which was significantly higher than that of the control group (9.4%) and the isotype control group (10.0%), similar to the LPS-positive control group (51.7%). Additionally, it was confirmed that the expression of M1-specific surface markers CD80 and CD197 also increased in the antibody-treated group compared to the control group (Fig. 11).

[0214] These results show that the anti-CD300c monoclonal antibody induces PBMC-derived primary macrophages into the M1 phenotype, confirming its potential for use in the fields of immune modulation and anticancer immunotherapy.

[0215]

[0216] Example 1.12. Measurement of expression of M1 macrophage markers secreted from primary macrophages

[0217] The concentrations of TNF-α, IL-1β, and IL-8 in the culture supernatant of primary macrophages were measured using the Quantikine ELISA kit system (R&D Systems, Minneapolis, MN). Primary macrophages were seeded into 24-well culture plates at a cell density of 1 × 10⁶ cells per well, and anti-CD300c monoclonal antibody was administered along with an LPS-positive control to induce M1 macrophage activation. After 48 hours of culture, the supernatant was collected, and the concentrations of each cytokine were measured using the Quantikine ELISA kit according to the manufacturer's instructions; the results are shown in Figure 12.

[0218] As a result, the secretion of TNF-α, IL-1β, and IL-8 was significantly increased in the group treated with the anti-CD300c monoclonal antibody. These results indicate that the anti-CD300c monoclonal antibody promotes the M1-type functional differentiation of primary macrophages, thereby enhancing the production of inflammatory cytokines.

[0219]

[0220]

[0221] Example 1.13 Confirmation of the activating effects of anti-CD300c monoclonal antibody on MAPK (mitogen-activated protein kinase) and NF-κB (nuclear factor κB) signaling pathways

[0222] Western blot analysis was performed to identify the signaling pathway by which the anti-CD300c monoclonal antibody induces M1 differentiation in primary macrophages. Cells were treated with lysis buffer on ice for 10 minutes to obtain lysates, proteins were separated via SDS-PAGE, and transferred to a nitrocellulose membrane. The membrane was blocked in a 5% skim milk solution for 30 minutes and then reacted with primary and secondary antibodies including α-p38, α-phospho-p38, α-ERK1 / 2, α-phospho-ERK1 / 2, α-SAPK / JNK, α-phospho-SAPK / JNK, α-phospho-NF-κB, and HRP-conjugated anti-rabbit IgG. Subsequently, chemiluminescence signals were detected using an ECL reagent (Thermo Fisher Scientific), and the intensity of the Western blot bands was quantified using ImageJ software (n=3). The results are shown in Figure 13.

[0223] As a result, it was confirmed that the phosphorylation of ERK and p38 (p-ERK, p-p38) and the NF-κB p65 phosphorylation subunit increased rapidly within 15 to 30 minutes after antibody treatment.

[0224] Specifically, the phosphorylation forms of p38, p44 / 42 MAPK, JNK, and NF-κB increased by approximately 1.5-, 7-, 10-, and 3-fold, respectively. This suggests that the anti-CD300c monoclonal antibody activates the MAPK and NF-κB signaling pathways to induce M1 differentiation of primary macrophages.

[0225]

[0226] Example 2. Confirmation of the effect of anti-CD300c monoclonal antibody in increasing M1 macrophage differentiation and reducing tumor size in a mouse colorectal cancer model

[0227] Example 2.1. Confirmation of the in vivo cancer growth inhibitory effect of anti-CD300c monoclonal antibody

[0228] To evaluate the anticancer effect of the anti-CD300c monoclonal antibody under in vivo conditions, a syngeneic mouse model was established by subcutaneously injecting colorectal cancer cell lines (CT26, 2 × 10⁵ cells) into 7-8 week old BALB / c mice. All animal rearing and experiments were performed in a specific pathogen-free (SPF) environment.

[0229] When the tumor size exceeded 50 mm³, mice were injected with an anti-CD300c monoclonal antibody or an anti-PD-1 antibody (clone J43; BioXCell, Lebanon, NH) a total of four times on days 0, 3, 6, and 9. As shown in Figure 14, tumor growth was inhibited by approximately 30% in the anti-PD-1 treatment group compared to the control group, and 42% inhibition was observed in the anti-CD300c monoclonal antibody treatment group alone.

[0230] These results confirm that the anti-CD300c monoclonal antibody exhibits a significant effect on inhibiting tumor growth in an allogeneic colorectal cancer mouse model.

[0231]

[0232] Example 2.2. Tumor-bound expression of M1 macrophages in mice administered anti-CD300c monoclonal antibody or anti-PD-1 monoclonal antibody

[0233] The acquired tumor tissue was fixed with 1% PFA, dehydrated overnight using 20% ​​sucrose solution, and frozen (Leica, Wetzlar, Germany). The frozen tissue was sectioned to a thickness of 50 μm, permeated with 0.3% PBST, and non-specific binding was blocked by adding 5% normal goat serum to the PBST. Subsequently, the samples were incubated overnight with anti-iNOS (Abcam, Cambridge, UK) and anti-CD206 (Invitrogen). After three washes, secondary antibody treatment with FITC-conjugated anti-rabbit IgG (Jackson ImmunoResearch, West Grove, PA) and Cy3-conjugated anti-rat IgG (Jackson ImmunoResearch) was performed at room temperature for 2 hours. Finally, the sample was mounted on a mounting medium (DAKO, Carpinteria, CA), and images were acquired using an LSM 880 microscope (Carl Zeiss, Dublin, CA), and the results are shown in Fig. 15.

[0234] Analysis results showed that while M1 macrophages increased slightly in the anti-PD-1 antibody treatment group compared to the control group, a significant increase in M1 macrophages was observed in the anti-CD300c monoclonal antibody treatment group.

[0235] These results demonstrate that the anti-CD300c monoclonal antibody can effectively promote M1 macrophage differentiation compared to existing immunotherapies.

[0236]

[0237] Example 2.3. Confirmation of the effect of anti-CD300c monoclonal antibody on increasing M1 macrophages

[0238] To evaluate in vivo M1 / M2 macrophage polarization, tumors harvested from mice administered anti-CD300c monoclonal antibodies were cleaved and incubated at 37°C for 1 hour in digestion buffer containing collagenase D (Roche, Grenzach-Wyhlen, Germany) and DNase I (Roche). The resulting cell suspension was filtered through a cell filter (Corning, Corning, NY), treated at room temperature for 3 minutes to remove erythrocytes and cell clumps, washed with FACS buffer, and filtered through a nylon mesh. Cells were treated with Fixable Viability Dye eFluor™ 450 (Invitrogen, Carlsbad, CA) on ice for 30 minutes, followed by staining with CD11b (M1 / 70, Invitrogen) and F4 / 80 (BM8, Invitrogen) antibodies on ice for 30 minutes. Stained cells were analyzed using CytoFLEX (Beckman Coulter), and the data were processed using FlowJo version 10 (Tree Star, Ashland, OR). The results are shown in Figure 16.

[0239] Analysis results showed that the anti-CD300c monoclonal antibody significantly increased differentiation into M1 macrophages. It was confirmed that these increased M1 macrophages activated various immune cells, particularly cytotoxic T cells, at high concentrations, contributing to tumor growth inhibition and the reprogramming of the tumor microenvironment (TME).

[0240]

[0241] Example 3. Anticancer effect in LLC model upon treatment with anti-CD300c antibody

[0242] Example 3.1 Confirmation of the tumor growth inhibitory effect of anti-CD300c antibody

[0243] The anticancer effect of the anti-CD300c antibody in the Lewis-lung carcinoma (LLC) model was confirmed by the inhibition of tumor growth. Luciferase-expressing LLC cells were introduced with the firefly luciferase gene (luc2) using a lentiviral vector to enable bioluminescence for in vivo imaging.

[0244] Male wild-type C57BL / 6J mice (6–8 weeks old) were purchased from DBL (Chungbuk, South Korea). To create a mouse lung cancer orthotopic model, 2×10⁶ mice were placed in Matrigel matrix (Corning, NY, USA). 4 A 30 μL solution of suspended LLC-Luc cells was prepared. The left dorsal side of the mouse, from which all hair had been removed using Nair, was wiped three times with ethanol and Betadine to prepare for sterile surgery. Using scissors, a small horizontal incision was made to incise the skin, and the underlying fat layer was carefully incised. 2×10⁶ 4 A 30 μL suspension of LLC-Luc cells was injected into the injection site on the left lung (between the 5th and 6th ribs) using an insulin syringe. Subsequently, the skin was sutured again using 3M Vetbond tissue adhesive. Three days after inoculation, anti-CD300c monoclonal antibodies (5 mg / kg and 10 mg / kg) were injected into the peritoneal cavity of the mice, and changes in tumor size were measured. For measurement, D-luciferin (150 mg / kg) was injected into the peritoneal cavity of the mice, and imaging was performed 10 minutes later using an in vivo imaging system (Berthold Technologies). After four injections of the drug, mouse tumor growth was detected using in vivo bioluminescence, and lung tissue was collected.

[0245] Tumor growth was investigated in the LLC orthotopic model using an in vivo bioluminescence imaging system (NightOWL II; Berthold Technologies GmbH, Wildbad, Germany) at the end of drug administration. Luminescence signals were detected 15 minutes after the injection of D-luciferin (BioVision, Milpitas, CA, USA) (4 mg per mouse) with an exposure time of 0.1 seconds and 4 × 4 binning. Photon energy and tumor area were analyzed using IndiGO software (Berthold Technologies GmbH).

[0246] To investigate the effect of anti-CD300C monoclonal antibody treatment on tumor growth in a non-small cell lung cancer (NSCLC) model, an orthotopic NSCLC mouse model was established and anti-CD300C monoclonal antibody was administered at a dose of 5 mg / kg or 10 mg / kg.

[0247] As a result, as shown in Figure 17, tumor size was significantly reduced in both anti-CD300C monoclonal antibody administration groups compared to the PBS-administered control group. Tumor size tended to be smaller in the 10 mg / kg group than in the 5 mg / kg group.

[0248] From these results, it can be seen that treatment with anti-CD300C monoclonal antibodies significantly inhibits tumor growth in an NSCLC orthotopic model.

[0249]

[0250] Example 3.2 Confirmation of the effect of anti-CD300c antibody on viability

[0251] Male wild-type C57BL / 6J mice (6–8 weeks old) were purchased from DBL (Chungbuk, South Korea). To construct a mouse lung cancer subcutaneous model, 1×10⁶ were placed in a Matrigel matrix (Corning, NY, USA). 5A 100 μL solution of suspended LLC cells was prepared and subcutaneously inoculated into the right flank. When the tumor volume reached 50 mm³, the mice were intravenously injected with an anti-CD300C monoclonal antibody (10 mg / kg) every 3 days. After inoculation, when the tumor size reached a maximum diameter of 1.5 cm, the mice were sacrificed. The analysis results are shown in Figure 18.

[0252] Mice administered with anti-CD300C monoclonal antibody (10 mg / kg) showed a significantly increased survival rate compared to the PBS-treated control group, with median survival being 19 and 26 days, respectively. The results suggest that anti-CD300C monoclonal antibody treatment reduces tumor growth and extends survival time in an NSCLC model.

[0253] The group administered with the anti-CD300C monoclonal antibody (10 mg / kg) showed a significantly increased survival rate compared to the PBS control group, confirming that the anti-CD300C monoclonal antibody contributes not only to tumor suppression but also to the extension of survival in the NSCLC model.

[0254]

[0255] Example 3.3 Confirmation of the effect of anti-CD300c antibody on tumor proliferation

[0256] Lung tissue from tumor-bearing mice was fixed in 10% neutral buffered formalin and sectioned to a thickness of 5 μm. Antigen recovery was performed by placing the sections in 10 mM sodium citrate buffer (pH 6.0) and heating them in a microwave. After washing with PBS, the sections were blocked for 30 minutes with normal serum provided in the VECTASTAIN® ABC-HRP kit (PK-6101, Vector Laboratories, Newark, CA, USA). The sections were then incubated overnight at 4°C with rabbit polyclonal anti-PCNA antibody (1:200, Santa Cruz Biotechnology, Dallas, TX, USA). After washing with PBS, a biotinylated secondary antibody was applied, and the sections were incubated with the VECTASTAIN® ABC reagent. The signal was visualized using the DAB peroxidase substrate kit (SK-4100, Vector Laboratories). Slides were washed with tap water, counterstained with hematoxylin, dehydrated, and fixed with coverslips. Tissue slides were imaged at 20x magnification using an optical microscope (Olympus, Tokyo, Japan). Three fields were randomly selected, and PCNA-positive cells were quantified using QuPath software.

[0257] To determine whether anti-CD300C monoclonal antibodies affect tumor cell proliferation in non-small cell lung cancer (NSCLC), the expression of PCNA, a proliferation marker, in tumor tissue was examined using immunohistochemical staining, and the results are shown in Figure 19. PCNA expression was reduced in the anti-CD300C monoclonal antibody treatment group compared to the PBS treatment group. Along with the reduction in tumor growth, PCNA-positive cells were also significantly reduced in the anti-CD300C monoclonal antibody treatment group. These results suggest that anti-CD300C monoclonal antibodies inhibit tumor growth and proliferation.

[0258]

[0259] Example 3.4 Confirmation of the effect of anti-CD300c antibody on M1 macrophage differentiation

[0260] Lung tissue was collected from mice sacrificed for flow cytometry analysis and placed in MACS C tubes (Miltenyi Biotec, Auburn, CA, USA) containing Collagenase D (1 mg / mL; Sigma-Aldrich, St. Louis, MO, USA) and DNase1 (1 mg / mL; Sigma-Aldrich) in serum-free medium. The tissue was dissociated using a MACS dissociator (Miltenyi Biotec) and digested in a shaking incubator at 37°C for 25 minutes. The sample was then filtered using a 40 μm cell filter (Corning Incorporated, Corning, NY, USA) to obtain a single-cell suspension. Red blood cells (RBCs) were lysed in 1× RBC lysis buffer (Invitrogen, Carlsbad, CA, USA) at room temperature for 5 minutes. The cells were washed and resuspended in BD Pharmingen™ Stain Buffer (BD bioscience, San Jose, CA, USA). Cells were stained using antibodies at 4°C for 45 minutes. For bone marrow cell identification, the following antibodies were purchased from BD Biosciences or Biolegend (San Diego, CA, USA): mouse CD45-FITC, CD11b-PECy7, CD86-BV786, CD206-APC, F4 / 80-PE, CD11c-APC-Cy7; for lymphocyte infiltration identification, mouse CD45-APC-Cy7, CD8-PE-Cy7, CD4-BB700, CD25-BV421, Granzyme B-APC. For intracellular staining, cells were treated with 1x Fix and Permeation Buffer (BD Biosciences) for 30 minutes. Single-cell suspensions were washed and stained with Granzyme B. Data were collected using the BD FACSlyric™ (BD Biosciences) flow cytometry system and analyzed using BD FACSuite software (BD Biosciences).

[0261] The anti-CD300C monoclonal antibody, a CD300C monoclonal antibody, induced repolarized M1 macrophages from monocytes (20). To determine whether the anti-CD300C monoclonal antibody affects the regulation of macrophage populations within the tumor microenvironment (TME) of non-small cell lung cancer (NSCLC), macrophage populations were analyzed using a flow cytometer in lung tissue from mice with tumors. The anti-CD300C monoclonal antibody treatment group showed a significant increase in the M1 macrophage population compared to the PBS group.

[0262]

[0263] Example 3.5 Confirmation of the effect of anti-CD300c antibody on M1 macrophage differentiation

[0264] Lung tissue was collected from mice sacrificed for flow cytometry analysis and placed in MACS C tubes (Miltenyi Biotec, Auburn, CA, USA) containing Collagenase D (1 mg / mL; Sigma-Aldrich, St. Louis, MO, USA) and DNase1 (1 mg / mL; Sigma-Aldrich) in serum-free medium. The tissue was dissociated using a MACS dissociator (Miltenyi Biotec) and digested in a shaking incubator at 37°C for 25 minutes. The sample was then filtered using a 40 μm cell filter (Corning Incorporated, Corning, NY, USA) to obtain a single-cell suspension. Red blood cells (RBCs) were lysed in 1× RBC lysis buffer (Invitrogen, Carlsbad, CA, USA) at room temperature for 5 minutes. The cells were washed and resuspended in BD Pharmingen™ Stain Buffer (BD bioscience, San Jose, CA, USA). Cells were stained using antibodies at 4°C for 45 minutes. For bone marrow cell identification, the following antibodies were purchased from BD Biosciences or Biolegend (San Diego, CA, USA): mouse CD45-FITC, CD11b-PECy7, CD86-BV786, CD206-APC, F4 / 80-PE, CD11c-APC-Cy7; for lymphocyte infiltration identification, mouse CD45-APC-Cy7, CD8-PE-Cy7, CD4-BB700, CD25-BV421, Granzyme B-APC. For intracellular staining, cells were treated with 1x Fix and Permeation Buffer (BD Biosciences) for 30 minutes. Single-cell suspensions were washed and stained with Granzyme B. Data were collected using the BD FACSlyric™ (BD Biosciences) flow cytometry system and analyzed using BD FACSuite software (BD Biosciences).

[0265] To investigate the effects of anti-CD300C monoclonal antibody therapy on lymphocyte cell populations, flow cytometry was performed on lung tissue from non-small cell lung cancer (NSCLC) carrier mice.

[0266] The analysis protocol of this embodiment is shown in FIG. 23, and the analysis results according thereto are shown in FIG. 24.

[0267] A significant decrease in regulatory T cells (Treg) was observed in the anti-CD300C monoclonal antibody treatment group compared to the PBS control group. Additionally, anti-CD300C monoclonal antibody treatment significantly increased the CD8+ T cell population. These results suggest that the anti-CD300C monoclonal antibody may contribute to alleviating the immunosuppression of the TME in conjunction with cytotoxic CD8+ T cells.

[0268]

[0269] Example 3.6 Confirmation of the effect of anti-CD300c antibody on M1 macrophage differentiation

[0270] Total RNA was isolated from lung tissue of tumor-bearing mice using the easy-BLUE RNA extraction kit (iNtRON Biotechnology, Seongnam, South Korea). cDNA was synthesized using Cyclescript reverse transcriptase (Bioneer, Daejeon, South Korea) according to the manufacturer's instructions. Real-time PCR was performed using the CFX connect real-time PCR system (Bio-Rad Laboratories, Hercules, CA, USA) and the SensiFAST SYBR no-Rox kit (Bioline, London, UK). The expression levels of target mRNA were normalized to the expression levels of the housekeeping gene, mouse GAPDH. All fold changes were expressed relative to the PBS group. Each reaction was performed in duplicate.

[0271] To evaluate the immunomodulatory effects of anti-CD300C monoclonal antibody therapy in the tumor microenvironment of non-small cell lung cancer (NSCLC), the expression levels of immune-related genes were analyzed using qRT-PCR.

[0272] The analysis protocol of the present embodiment is shown in FIG. 23, and the analysis results according thereto are shown in FIG. 25 to 26.

[0273] The expression of M1 macrophage markers Nos2 and Cd86 was significantly increased in the anti-CD300C monoclonal antibody treatment group compared to the PBS group, which promotes inflammatory macrophage activation. In contrast, the expression levels of M2 markers such as Arg1 and Mrc1 did not show significant changes. Anti-CD300C monoclonal antibody treatment also upregulated the expression of inflammatory cytokines Tnfα and Il1β. Notably, the expression of immunosuppressive markers, including Foxp3, Ctla4, Il10, and Vegfa, was significantly reduced in the anti-CD300C monoclonal antibody treatment group. These results suggest that anti-CD300C monoclonal antibodies alleviate immunosuppression within the non-small cell lung cancer tumor microenvironment and promote anti-tumor immune responses.

[0274]

[0275] Example 4. Confirmation of the breast cancer inhibitory effect of anti-CD300C monoclonal antibody in vivo

[0276] Example 4.1. Confirmation of In Vivo Anticancer Effect of Anti-CD300c Monoclonal Antibody

[0277] In order to confirm the anticancer effect according to the route of administration and dosage when the anti-CD300c monoclonal antibody was administered to a solid tumor model, 1x10 breast cancer cell lines (4T1) 5 A mouse allograft tumor model was created by transplanting a dog into a BALB / c mouse via subcutaneous injection.

[0278] Subsequently, the anticancer effect of the anti-CD300c monoclonal antibody was observed in a TNBC (triple-negative breast cancer) mouse model, and the results are shown in Figure 27. As can be seen in Figure 27, a reduction in tumor size was confirmed in both the intraperitoneal and intravenous administration groups of the anti-CD300C monoclonal antibody. When the anti-CD300C monoclonal antibody was administered intraperitoneally, significance was confirmed compared to the control group at a dose of 10 mg / kg, and significance was observed in all administration groups when administered intravenously. The tumor suppression effect caused by the administration of the anti-CD300C monoclonal antibody showed dose dependence, increasing with the administered dose, and a higher anticancer effect was confirmed with intravenous administration than with intraperitoneal administration.

[0279]

[0280] Example 5. Confirmation of genetic changes induced by anti-CD300C monoclonal antibody

[0281] Example 5.1. Nanostring gene expression profiling

[0282] The following experiment was conducted to investigate the anticancer efficacy of the anti-CD300c monoclonal antibody (CL7). RNA was extracted and purified from resected tumors in CT26 colorectal cancer model mice, and the expression levels of various immune-related genes were measured. Gene expression values ​​were based on log2-normalized Nanostring gene expression profiling data.

[0283] Analysis results confirmed that the expression level of IL-7 decreased when treated with the anti-CD300C monoclonal antibody.

[0284] Probe Namefold changelog2FCIl70.7897-0.3407Il7r1.19740.2599

[0285] Based on the above results, the inventors intended to complementarily enhance the survival and long-term maintenance capabilities of activated T cells by developing an anti-CD300C monoclonal antibody-IL-7 fusion protein, as the reduction in gene expression levels following treatment with the anti-CD300C monoclonal antibody may limit the support of specific helper cell populations. Through this, the inventors aim to structurally amplify the anti-cancer response based on T cell activation induced by treatment with the anti-CD300C monoclonal antibody alone and further enhance the immune effect in the tumor microenvironment (TME).

[0286] Example 5.2 Confirmation of IL-7 expression pattern upon treatment with anti-CD300C monoclonal antibody

[0287] To investigate changes in IL-7 expression following treatment with anti-CD300c monoclonal antibodies, IL-7 expression levels were analyzed in six cell lines, including pMonocyte, THP-1, A549, Jurkat, HCT116, and MDA-MB-231, after treatment with anti-CD300c monoclonal antibodies. Human Cytokine / Chemokine / Growth Factor Panel A (Merck Millipore, # HCYTA-60K-PX48) was used for the quantification of IL-7.

[0288] Peripheral Blood Mononuclear Cells (PBMCs) were isolated from whole blood collected from healthy humans, and human-derived Primary Monocytes were purified and used. Primary Monocytes were placed at a density of 1 × 10⁶ per well in a 24-well plate. 6 After seeding with cells, each well was treated alone with anti-CD300C monoclonal antibody (10 μg / mL) and incubated for 48 hours. THP-1 was 1.5 × 10⁶ per well in a 96-well plate. 4After equal seeding of cells, the anti-CD300C monoclonal antibody was added alone to isolated wells and cultured for 48 hours. A549, Jurkat, HCT116, and MDA-MB-231 were also plated at 3 × 10⁶ cells per well in a 96-well plate. 4 Cells were inoculated, and each of the two cell lines was treated with an anti-CD300C monoclonal antibody (10 μg / mL) alone and cultured for 48 hours.

[0289] After 48 hours of incubation, the supernatant obtained from each well was vortex-homogenized for use as a sample, and then centrifuged at 10,000×g for 2 minutes under refrigerated conditions to recover only the supernatant. All samples were used for analysis without dilution. The samples and bead-antibody mixtures were incubated overnight at 4°C, while standards, blanks, and controls were treated under the same conditions as the samples using the assay buffer included in the kit. All experimental procedures were performed in strict adherence to the kit's protocol.

[0290] After the reaction was complete, the fluorescence amount of PE (phycoerythrin) bound to each bead was measured using a Luminex instrument (Luminex, Austin, TX, USA), and the median fluorescence intensity (MFI) value was obtained. Each analyte selectively binds to a capture antibody bound to a bead assigned a unique number, and a signal was generated in a sandwich manner with the detection antibody and streptavidin-PE.

[0291] The standard curve was generated using the best-fit algorithm of MasterPlex QT 2010 (MiraiBio, Hitachi, CA, USA) software. The software automatically selected the curve with the highest calculated R-squared value and established the optimal standard curve by removing or adjusting standard points where the concentration recovery rate deviated abnormally. Based on this, the IL-7 concentration of each sample was investigated.

[0292]

[0293] Primary MonocyteCytokineControlCL7 10Fold-decrease(Con. VS CL7)(pg / ml)IL-70.970.80.82THP-1CytokineControlCL7 10Fold-decrease(Con. VS CL7)(pg / ml)IL-71.070.550.51A549CytokineControlCL7 10Fold-decrease(Con. VS CL7)(pg / ml)IL-73.322.790.84MDA-MB-231CytokineControlCL7 10Fold-decrease(Con. VS CL7)(pg / ml)IL-741.524.80.60

[0294] Analysis results showed that IL-7 expression decreased in primary monocytes upon treatment with the anti-CD300C monoclonal antibody. Since these changes in cytokine expression were observed in human-derived primary monocytes, the results are similar to those found in the human body. Additionally, IL-7 expression decreased in THP-1 cells, A549 cells, and MDA-MB-231 cells upon treatment with the anti-CD300C monoclonal antibody.

[0295] IL-7 is known as a T cell activation cytokine. Since IL-7 generally tended to decrease as described above upon treatment with the anti-CD300C monoclonal antibody, there was a need to provide a linker-linked fusion protein to compensate for the reduced IL-7.

[0296]

[0297] Based on these results, the applicants intended to develop a fusion protein based on an anti-CD300C monoclonal antibody to exhibit a more potent anticancer effect through a synergistic effect between the two immune cell populations, combining the macrophage activation effect of the anti-CD300C monoclonal antibody with the T cell activation effect of IL-7.

[0298]

[0299] Example 5.3. Confirmation of changes in RNA expression levels

[0300] RNA sequencing analysis was performed to evaluate the anticancer efficacy of the anti-CD300C monoclonal antibody, and total RNA extracted from the samples was aligned and quantified using a standard pipeline. Gene expression values ​​were calculated based on log₂-normalized RNA-Seq data. The analysis results confirmed that treatment with the anti-CD300C monoclonal antibody increased or decreased the expression levels of various genes, including TP73, CASP8, and RAD51. TP73 is a tumor suppressor gene similar to p53 that inhibits the abnormal proliferation of cancer cells by inducing apoptosis and cell cycle arrest under cellular stress. CASP8 is a key protein that regulates the early stages of the apoptosis pathway and contributes to the anticancer effect by activating endogenous and exogenous apoptotic signals that induce cancer cells to die spontaneously. RAD51 is a gene involved in DNA damage repair that performs a tumor suppressor role by maintaining genomic stability and reducing the likelihood of tumor development caused by mutations. AGK, ARSD, CELSR3, MGST1, GAB2, FOXP3, HOMER3, NNAT, TNK2, GPC1, LIPE, IL12RB2, ITGAE, MAPRE3, PPM1F, PTK6, ABCD1, HCFC1R1, PDGFRL, CALB1, TRPS1, SPAG1, PLPPR2, ARRDC2, TAX1BP1, MAPK8, UBE2S, RECQL5, ALOX12, DHRS7B, DUSP16, ASF1A, MDN1, IK, LMNB1, OPRD1, ASH1L, CD160, PIK3R3, DARS2, UBN1, PROX2, KBTBD7, TGFBI, ACO1, DDX54, PAIP2B, GPR18, OPA3, including these genes, by treatment with anti-CD300C monoclonal antibody FAM78A, ARHGAP22, MPDU1, THEM6, GJA9, SLC6A6, MACROD1, ZFC3H1, CAMK1, NAV1, DMTF1, CEP350, RAC1, YME1L1, FPGS,THBS1, CYP1B1, EMILIN1, BMP2K, CCDC65, LINS1, RMC1, CCDC97, AZIN2, HDGF, ABCG8, SHPRH, CSGALNACT1, FBXO10, C11orf49, FADS1, ROM1, FCGR1A, MIA2, UBC, ITPR1, KCNJ1, DCLRE1C, ATP5PF, MMS19, PAXBP1, IFNAR2, GNG3, TRIM17, ANXA5, GJB7, LETM2, CYBB, NSD1, CLMN, DCHS1, THAP9, SMIM4, MGAT2, COG7, SEMA4C, ZFPM2, TRIM8, LDHAL6B, TADA2B, FAM222B, RXFP4, LRFN4, C9orf131, ZDHHC24, DENND4A, PDZK1, TBC1D10C, ANKLE2, DEAF1, KCNA3, SOX12, FAM87B, ODF3B, PDE4DIP, MAF, TIGD5, WDFY3-AS2, ZNF707, MRPS16, TMEM198B, C11orf54, SRPRA, ALDH1A3, TCTE3, RPS27L, ZSWIM9, IFIT1, ZNF749, GOLGA8R, PCDHB13, SEMA4D, C15orf62, NEMP2, NLGN3, TSC22D2, MAML3, AC068631.1, ZXDA, PRC1, RPL12P1, TRIM39, INSYN2B, AL590399.1, ATXN2, C12orf73, ANKUB1, DIO1, LCAT, PIGCP1, LINC01521, LIPE-AS1, ZSWIM8, SRSF9P1, SMTNL1, RPS3AP24, SEC13P1, AC063976.2, PFN1P4, AC005281.1, AC007679.2, LINC00937, AL354836.1, AL354977.1, CUTALP, AL136988.2, AL020996.1, ARMCX3-AS1, AC092802.3, PGA4, CXCR2P1, LINC00102, AL021937.1, DSTNP1, AL137856.1, RNF2P1,EP300-AS1, AL133230.2, AL158166.2, AC010733.1, AC000078.1, LINC01681, AC116366.2, AC012618.2, ATP5PDP4, AC010978.1, NPM1P26, MYCL-AS1, CBR3-AS1, ZNF503-AS2, LINC01637, AC008937.2, AC004893.1, TNFRSF14-AS1, TLR9, AC012358.4, PTPRG-AS1, AC244197.3, AP001062.3, AC087752.1, FLNB-AS1, H2AZ1-DT, AP000787.1, AC022075.1, CEBPA, LACTB2-AS1, FAM13A-AS1, AL033397.2, HS3ST5, AP006623.1, AC106865.1, AC021087.2, AC020661.1, AC106760.1, WDFY3-AS1, AC005280.1, HAUS1P1, AC092611.1, AC025442.1, TNFRSF10A-AS1, AC105206.2, CD44-AS1, SMIM3, RRN3P3, AC073896.3, AC020656.1, AC011611.5, AC025034.1, AC078778.1, RAP1AP, AL442663.3, AC087286.3, AC066613.2, AC023906.4, LCMT1-AS2, AC141586.2, AL359258.2, ADPGK-AS1, AC016597.2, AC007728.2, CR936218.1, AC015563.3, AC020558.2, AP000919.2, AC100830.3, ZNF850, AC004490.1, AC011477.2, AC006116.11, AC063977.6, AC008760.1, AC090181.2, AC090241.2, AC174071.1, NRBF2P5, H3P37, AL445435.1, AL158163.1, HNRNPA3P9, HTATSF1P2, AC008937.3, NFYC-AS1, AC087752.4, ZSWIM8-AS1,AC253536.6, AC017083.3, AL139339.2, AC084824.4, AL139260.3, AC010503.5, AC084824.5, AL121772.3, AC018926.3, CISD3, AL031320.2, AC090181.3, AC233300.1, AL662907.2, AC008894.3, AC100788.2, Z99129.4, AC011815.3, HELLPAR, SEPSECS-AS1, HSFX3, AC092053.4, AC009554.2, AL162586.2, Z80897.1, AC011416.4, AP002748.6, AL360020.1, AJ011931.2, AC092881.2, AC106865.2, AL136379.1, CFTR, SARM1, ST3GAL1, GABRA1, AGPAT4, PNPLA6, USP2, MTREX, MED17, RETSAT, MRTO4, KMT2C, MTMR1, CASP8. ARFGEF1, PANX2, TUBA3D, GNB1, AK6, CETP, CAPN3, DHPS, DDX17, SUN2, STK4, ARFRP1, APMAP, SUV39H1, TAZ, PGK1, TSNAXIP1, GSPT1, ATP13A1, BUD31, PLOD3, KCNT1, TBC1D12, UGDH, DNAJC4, PDHX, CAMKK2, GPN3, NAA25, NOP2, SERINC1, VEGFA, CERT1, PPWD1, ABHD14B, IL1A, SRSF7, PDC, UCHL5, CTBS, PPP1R8, TXNDC12, ADGB, TNFAIP3, ZNF410, INSL6, TRIM25, NCOA3, USP22, XPO5, RREB1, NUP85, PAX8, F2RL3, SPCS3, AANAT, CEP85, MPP1, ZNF428, EMILIN2, GPALPP1, SPX, ARGLU1, CTSL, ITGA7, LTV1, CD164, CSH1, CREBZF, PAQR5, GCHFR, ATRAID, POC1B, MTMR6,CBLN3, NOB1, ARSG, ZNF750, ZNF473, PIGM, MCL1, TPM3, UBAP2L,CTDSP1, TM4SF19, SPATA9, FAM193B, ZNF182, EIF3H, SLC39A4, ADIRF, PTPRJ, ARFGAP2, CCDC81, P4HA3, CABLES2, C4orf33, GUF1, AHCTF1, TRAPPC8, C4orf19, PI4K2A, HK2, CLDND2, FDPS, LMNA, LY6E, SYCE2, OXER1, CTSS, NIPAL1, SENP2, EXO5, SGMS2, ​​PGRMC2, ABCE1, NDUFAF2, CTHRC1, SLC25A32, DCAF13,CPSF2, C1QL3, ARIH1, TRIM44, CYB5R2, TRUB2, CDT1, TP53I13, ZNF526, GLOD4, MFSD3, FILIP1L, SLC50A1, DRD5, TMEM266, GUSB, ALCAM, POLR1C, ZIK1, GPHN, CARNS1, STAG3L3, RPL15, RGMA, CMTM4, XPOT, BTBD6, FAM43A, RGPD2, TMEM179B, DRG1, KCNIP4, WWOX, TMEM222, RPS19BP1, CARD9, NUTM2B, RRP7A, AL031577.1, YRDC, SLC22A4, SPTAN1, ZNF181, SSBP3-AS1, ARHGAP11A, POLH-AS1, AC105020.1, XAGE1B, HLA-J, FOXD4L5, IFITM5, ELOA2, LINC01089, AL590762.1, AC104117.1, SEPHS1P6, GANC, APTR, AC141586.1, KRT18P12, AL133406.1, GAS8-AS1ZNF880, VPS52, AL360093.1, AC091799.1, WARS2-IT1, LINC00539, AC138028.2, LINC00863, AC008937.1, AC104134.1, AC007285.1, CNN2P1, LINC00392, AL513550.1, AC006460.1, AC023271.2, OR2A9P, BX664615.1, LYST-AS1, AC012507.1, SRGAP2-AS1, AL031666.1, KLF3-AS1, AL354733.2, DIRC3, TMEM253, MTRNR2L4, DFFBP1, CYP2T1P, CCDC188, AC118555.1, CPB2-AS1, APOA1-AS, AC009319.1, AC007750.1, SMG1P1, DGUOK-AS1, AC093110.1, ZDHHC4P1, BX470102.1, ETF1P2, GHRLOS, CORT, RBBP4P2, RPL23AP81, AC022973.2, AC134772.1, ODCP, GABPB1-AS1, AC009126.1, AC092658.1, AC008906.1, AC110373.1, AC020651.1, AC096564.2, AC138956.2, HADHAP1, AC010260.1, AC004707.2, AC025370.1, AC024451.3, SLC2A3P4, AC022973.4, AP003392.1, AP001107.6, AP003031.3, AP002990.1, AP003041.2, FDXACB1, ZNF350, AC034102.6, AL121603.2, HNRNPMP1, TPM1-AS, CERNA1, AC015712.2, AC012184.1, AC026471.1, AC004158.1, HNRNPA3P11, AC009093.3, AC090260.2, LINC00563, AC007216.2, AC093525.6, AC093249.6, MTCO1P28, AC130651.1, AC015853.1, MAGOH2P, AC098850.1, AC093484.2,AC011825.4, ARGFXP2, AC138207.5, TXNIP, AP001793.1, AC006213.3, AC005264.1, AC022154.1, AC008764.3, BNIP3P24, ZNF350-AS1, PTMAP11, AC022400.4, AL353807.4, BNIP3P11, AC087623.2, AC016394.3, AL022238.3, AL391834.1, AC084018.1, AL022324.3, AC018690.1, AC096586.2, NATD1, RCC1L, AC012150.2, AL031670.1, AC084824.6, AC069234.4, HERC2P2, AC006449.5, AC004812.2, FO681492.1, AL359693.1, AL358852.1, AC080112.4, HEIH, AC073130.3, AC073592.6, AC008739.5, AL845472.2, AC007342.7, AC120114.3, BTG3-AS1, AL358472.4, AC004381.4, AC234917.2, BMP8B-AS1, AL356776.2, AL139286.3, AC244250.4, AC016026.2, AC073578.5, AC027811.1, AL161662.1, AC009630.4, Z98886.1, AC004825.3, AL596442.4, AC139272.1, AL357874.3, GNPNAT1, SRP19, HSP90AA1, TMEM218, CHD2, RBM6, ALDH3B1, VSIG2, RNF19A, ARID1B, ROGDI, CLNS1A, BAZ2A, CAMSAP3, TP73, ZMPSTE24, JPH4, AGO1, LZTR1, FAM118A, PIGH, YY1, MRGBP, HM13, IL21R, RNF40, CSPP1, CDK6, GSDME, ANAPC15, ALDH2, CCN6, TTC1, GNPDA1, ICMT, RPL22, STXBP3, ERRFI1, PRDX6, YLPM1, NR2C1, MAPK8IP1,SERAC1, AL365205.1, SSR1, RPL23, THRA, PLEKHG3, GALR3, CBFA2T3, CYP2E1, UBE4B, ACSS2, SLF1, IER3IP1, CABLES1, ADAMTS8, SDS, TRAFD1, CCDC146, CD63, CTDSPL2, EAF1, ILDR1, CISD2, RPS3A, GINS4, LRRC27, EIF3M, NUDT22, SLC26A2, ATP2B2, DDX19B, JOSD2, TMEM183A, HMGB2, TIGD6, FBXO33, RPL36AL, NUDT5, CLEC1B, JCAD, STAT6, CNPY4, PDIA3, SEMA6B, ZNF232, ATXN2L, HINT1, HEXD, ZNF274, BSG, RAB40A, CENPS, LRRC57, IST1, NCF4-AS1, COPB2, POU3F2, GLDN, FAM166A, ZNF846, WDR5B, RPL37A, ZNF461, AL136038.1, RXRB, HLA-F, ZBTB10, AKR7L, MAP10, AL591135.1, DUX4L9, POT1-AS1, ARL2BPP10, RAD23BP3, AC005682.1, AL158834.2, AL031963.1, KCNQ5-AS1, LINC00689, AC026412.2, DDX3P1, AL773545.3, AL365273.1, SNHG6, MTND5P11, KRT18P25, HOXA-AS2, CASC19, BRK1, AC021092.2, AC034102.5, AL139022.1, LINC02367, AC006111.1, SPDYE9P, AC002398.1, AC011511.3, AP005131.2, ERFL, MAGIX, AC010336.6, AL670729.1, AC145423.1, MTDHP1, AC012645.4, H4C9, NR2E3, AC132938.5, AC012313.10, AC110285.6, AC006460.2, AL035078.4, FP236383.9, AL157769.1,AC009501.3, AC112236.3, AL035653.1, ARHGAP44, ELOA, RAD51, MCOLN3, ACADVL, FRY, CELSR1, ZNF638, SESN1, NUP188, SH3GLB1, PPIL2, HPS4, XBP1, ASCC2, SNW1, CHD8, JAG1, SLC1A5, ZNF574, HOXA2, TAF6, ZFAND5, CHRNE, HSPA8, UNC93B1, EIF4G2, GOLPH3, KPNA1, HYAL1, GNB4, STAT1, EHBP1, CHMP3, RLF, MPL, RPS25, RBBP6, CBX3, NCKAP1L, SLC12A4, MRS2, EMC1,SGCE, KCNC1, ACSBG2, ACTN4, TRAF7, XPO4, SRRM1, BIVM, AKIRIN2, RMDN3, IFT172, FBXO11, HNRNPD, YARS2, SRP14, SLC39A3, RPL13A, ENSA, LIPT1, PKP4, PLPP5, SAV1, TCF7L1, ANAPC1, PLA2R1, DBI, MIER3, FCSK, KIT, GAB3, RAVER1, ITIH3, ZNF496, WDR26, KIAA1841, HHIP, SLC29A4, ZNF507, OR2A20P, VCPIP1, DDIT3, ZNF575, ULK1, CASKIN2, ARIH2, SH2B1, PSTK, KCTD2, GINS3 TSEN54, UTY, COA3, WT1, MAFF, TET3, SMIM15, RPL14, C20orf204, PCBP2, NUDT16, ZNF536, AC131097.2, FP700111.1, PCOLCE-AS1, MRPL20-AS1, AC016394.2, MED4-AS1, AL359853.1, AL512625.2, AL589880.1, PPIAP40, RPS18, TMA7, RPS15AP12,U52111.1, H2BC15, ACSL3-AS1, AC137630.2, AC092809.5, LINC00649, LINC01352, CASTOR3, MIF, AL158847.1, AP002812.1, RPLP1P11, LINC02044, AC116535.2, AP000866.2, AC008581.1, AC013643.2, TMEM9B-AS1, AP001767.2, CEP95, AC090695.2, AC021231.2, AC022167.1, AC106820.3, AC087481.1, AC055855.2, BX255925.1, AC009053.4, PCDHB19P, AC004494.1, AC133552.4, AC015813.1, AC127024.3, AC138207.6, AC010761.5, RASSF5, ZNF790-AS1, AC010326.4, PCF11-AS1, NBPF14, AC026412.3, AC087623.3, AL031775.1, AC211476.4, AC097376.3, AL136295.6, AC004080.15, AL031595.2, AD000864.1, FP236383.3, GTF2IP12, AL021155.5, AL158212.5, AL358777.3, AC104066.5, AC073210.3, AC018695.9, AL139801.1, SMIM15, RPL14, C20orf204, PCBP2, NUDT16, ZNF536, XAGE1A, ZNF805, C17orf107, MT-RNR2, MARCKSL1P1, MPRIPP1, PPP3CB-AS1, AC005481.1, AC131097.2, FP700111.1, PCOLCE-AS1, MRPL20-AS1, AC016394.2, MED4-AS1, AL359853.1, AL512625.2, AL589880.1, PPIAP40, RPS18, TMA7, RPS15AP12, U52111.1, H2BC15, ACSL3-AS1, AC137630.2, AC092809.5, LINC00649, LINC01352,CASTOR3, MIF, AL158847.1, AP002812.1, RPLP1P11, LINC02044, AC116535.2, AP000866.2, AC008581.1, AC013643.2, TMEM9B-AS1, AP001767.2, CEP95, AC090695.2, AC021231.2, AC022167.1, AC106820.3, AC087481.1, AC055855.2, BX255925.1, AC009053.4, PCDHB19P, AC004494.1, AC133552.4, AC015813.1, AC127024.3, AC138207.6, Changes in the expression of AC010761.5, RASSF5, ZNF790-AS1, AC010326.4, PCF11-AS1, NBPF14, AC026412.3, AC087623.3, AL031775.1, AC211476.4, AC097376.3, AL136295.6, AC004080.15, AL031595.2, AD000864.1, FP236383.3, GTF2IP12, AL021155.5, AL158212.5, AL358777.3, AC104066.5, AC073210.3, AC018695.9, and AL139801.1 were confirmed.

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335] These observations suggest the possibility that regulating CD300c can influence the growth of cancer tissue by controlling the expression of various genes.

[0336]

[0337] Example 5.4. Correlation between CD300c and IL-7 in cancer

[0338] Previous analyses confirmed that various factors are associated with anti-CD300C monoclonal antibodies. Based on these results, the correlation between IL-7, a cytokine associated with T-cell activation, and CD300C was systematically investigated across various cancer types.

[0339] Correlation analysis between the CD300c and IL-7 genes was performed in November 2025 using the “Expression Correlation Analysis” module of the GEPIA2 web server (http: / gepia2.cancer-pku.cn / ). For the analysis, CD300c and IL-7 were entered into Gene A and Gene B, respectively, and Tumor-type-specific data from the TCGA dataset was selected from the tumor panel. Gene expression values ​​were based on RNA-Seq data normalized to log₂(TPM + 1), correlation evaluation was performed using Pearson correlation coefficients, and the results were visualized as scatter plots. The results are shown in Figures 28 and 29.

[0340] Analysis results showed that a total of 11 cancer types exhibited a high correlation in expression between CD300c and IL-7, including bladder urothelial carcinoma (bladder cancer), invasive breast cancer (breast cancer), head and neck squamous cell carcinoma (head and neck cancer), chromophorenal renal cell carcinoma and clear cell renal cell carcinoma (kidney cancer), hepatocellular carcinoma (liver cancer), ovarian serous cystadenocarcinoma (ovarian cancer), lung adenocarcinoma and lung squamous cell carcinoma (non-small cell lung cancer), prostate adenocarcinoma (prostate cancer), and cutaneous melanoma (skin cancer). These results suggest that CD300c and IL-7 are simultaneously expressed in specific cancer types and perform related roles.

[0341] As a high correlation between CD300c and IL-7 has been observed in various cancer types, the applicants based their findings on the previously confirmed anticancer effects in in vivo models of colorectal, breast, and lung cancer on the basis that the two substances perform functionally linked roles in these cancer types to exhibit synergistic effects regarding immune activation and anticancer effects within the tumor microenvironment, thereby forming the basis for the design of the fusion protein of the present invention.

[0342]

[0343] Example 6. Preparation of fusion protein CB701 containing anti-CD300c monoclonal antibody and IL-7

[0344] Example 6.1. Preparation of Fusion Protein CB701 Expression Vector

[0345] Based on the results of the above examples, the applicants devised a fusion protein (CB701) in which an anti-CD300C monoclonal antibody and the cytokine IL-7 are linked via a linker. In the present invention, a fusion protein CB701 was produced by stably combining the two proteins using a linker so that the anti-CD300C monoclonal antibody and IL-7 could maintain their respective biological activities (Fig. 30).

[0346] An expression vector was constructed by separating the heavy chain and light chain capable of expressing a fusion protein (hereinafter referred to as fusion protein CB701) containing the anti-CD300c monoclonal antibody and IL-7 through a codon optimization process using the nucleotide sequence of the anti-CD300c monoclonal antibody and the nucleotide sequence of IL-7.

[0347] More specifically, using the analyzed CDR sequence, genes were inserted into the pcDNA3.1 vector to express the heavy chain and light chain, respectively (Fig. 31).

[0348] The nucleotide and amino acid sequences encoding the heavy chain, light chain, and CDR, and the amino acid sequence of CD7 scFv-IL-17 are as follows.

[0349]

[0350] Sequence No. Name Sequence 1 Double-chain Amino Acid Sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSQGIFDIWGQGTLVTVSS 2 Double-chain Nucleotides SequenceGAGGTGCAGCTGTTGGAGTCTGGTGGAGGCTTGGTACAGCCTGGAGGTTCTCTTCGCCTCTCCTGTGCAGCCTCCGGATTCACTTTCAGCCGCTACGCAATGAGCTGGGTCAGACAGGCACCAGGTAAGGGACTGGAGTGGGTCTCTGCAATTAGCGGTAGCGGTGGTAGCACTTAC TACGCAGACAGCGTGAAGGGTCGCTTCACCATCTCACGCGACAACTCCAAGAACACCCTGTACCTGCAGATGAACAGCCTTCGCGCAGAGGACACTGCCGTGTATTACTGCGCACGTAGCAGCCAGGGTATCTTCGACATCTGGGGACAAGGTACTCTGGTCACTGTCTCCTCA3CDR1 Amino acid sequenceFTFSRYAMSWVR4CDR1 Nucleotide sequenceTTCACTTTCAGCCGCTACGCAATGAGCTGGGTCAGA5CDR2 Amino acid sequenceAISGSGGSTYYAD6CDR2 Nucleotide Sequence GCAATTAGCGGTAGCGGTGGTAGCACTTACTACGCAGAC7CDR3 Amino acid sequence YCARSSQGIFDIW8CDR3 Nucleotide sequence TACTGCGCACGTAGCAGCCAGGGTATCTTCGACATCTGG 9 Light chain amino acid sequence QSVLTQPPSASGTPGQRVTISCSGNNIGTRRVHWYQQLPDTAPKLLIYSKNNRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGPVFGGGTKLTVL 10 Light chain nucleotidesSequenceCAGTCTGTGCTGACTCAGCCACCTTCAGCATCTGGTACTCCAGGTCAGCGCGTCACCATCAGCTGCAGTGGTAACAATATCGGTACTAGACGCGTGCATTGGTATCAGCAACTCCCAGACACCGCTCCTAAGCTCCTGATTTACAGTAAGAACAACCGTCCTAG TGGTGTGCCTGATCGCTTTTCTGGGTCCAAGTCTGGCACCTCAGCCTCTCTGGCTATCAGTGGACTTCGCTCCGAGGACGAGGCTGACTATTACTGCGCAGCATGGGACGACAGCCTGAGCGGTCCTGTGTTCGGCGGTGGGACCAAACTGACCGTCCTA11CDR4 Amino acid sequenceCSGNNIGTRRVHW12CDR4 Nucleotide sequenceTGCAGTGGTAACAATATCGGTACTAGACGCGTGCATTGG13CDR5 Amino acid sequenceSKNNRPSGVP14CDR5 Nucleotide Sequence AGTAAGAACAACCGTCCTAGTGGTGTGCCT15CDR6 Amino acid sequence YCAAWDDSLSGPVF16CDR6 Nucleotide sequence TACTGCGCAGCATGGGACGACAGCCTGAGCGGTCCTGTGTTC39CL7 scFv-hIL7 Amino acid sequence EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSQGIFDIWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGNNIGTRRVHWYQQLPDTAPKLLIYSKNNRPSGVPDRFSGSKSGTSASLA ISGLRSEDEADYYCAAWDDSLSGPVFGGGTKLTVLGGGGSGGGGSGGGGSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEG MFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH40CL7scFv-mIL7 Amino Acids SequenceEVQLLESGGGLVQPGGSLRLSCAASGFTFSRYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY YCARSSQGIFDIWGQGTLVTVSSGGGGSGGGGSGGGGSQSVLTQPPSASGTPGQRVTISCSGNNIGTRRVHWYQQLPDTAPKLLIYSKNNRPSGVPD RFSGSKSGTSASLAISGLRSEDEADYYCAAWDDSLSGPVFGGGTKLTVLGGGGSGGGGSGGGGSECHIKDKEGKAYESVLMISIDELDKMTGTDSNCPNNEPNFFRKHVCDDTKEAAFLNRAARKLKQFLKMNISEEFNVHLLTVSQGTQTLVNCTSKEEKNVKEQKKNDACFLKRLLREIKTCWNKILKGSI

[0351] The prepared heavy and light chain expression vectors (7.5 μg each) were mixed with PEI (polyethylenimine) in a 1:1 mass ratio and transfected into ExpiCHO cells to induce the expression of the fusion protein (CB701). On day 10, the culture medium was centrifuged to remove the cells, and the culture medium was obtained. ExpiCHO cell culture and transfection were performed according to the ExpiCHO user manual. The obtained culture medium was filtered and then resuspended in a solution containing a mixture of 0.1 M NaH2PO4 and 0.1 M Na2HPO4 (pH 7.0). The resuspended solution was purified by affinity chromatography using Protein A beads (GE healthcare) and finally eluted using an elution buffer (Thermofisher binding buffer).

[0352] Example 6.2. Confirmation of Fusion Protein CB701 Expression

[0353] To verify the fusion protein CB701 prepared according to Example 6.1, 5 μg of purified CB701 was added to a reducing sample buffer and a non-reducing sample buffer, respectively, and electrophoresis was performed using pre-made SDS PAGE (Invitrogen), after which the protein was stained with Coomassie blue. The results of the reducing condition and the non-reducing condition are shown in Figure 32.

[0354] As shown in Fig. 32, it was confirmed that the high-purity fusion protein CB701 was produced and purified.

[0355]

[0356] Example 7. Confirmation of binding ability of anti-CD300c monoclonal antibody and fusion protein CB701

[0357] Example 7.1. Confirmation of binding affinity of fusion protein CB701 to CD300c (ELISA)

[0358] CD300c antigen (250 µg / mL, 11832-H08H, Sino Biological) or CD300a antigen was diluted to a concentration of 800 ng / well in coating buffer (0.1 M sodium carbonate, pH 9.0), 100 µL was added to each well of a 96-well microplate, and incubated overnight at 4°C. The next day, the plates were washed three times with 200 µL of PBST. Subsequently, 200 µL of blocking buffer (5% skim milk) was added, and the plates were blocked at room temperature for 1 hour. Anti-CD300c monoclonal antibody or fusion protein CB701 was serially diluted fourfold starting from a concentration of 2.5 µg / ml in PBS, 100 µL of each was added, and the mixture was reacted at room temperature for 1 hour to bind to the antigen. After the reaction, the unbound anti-CD300c monoclonal antibody or the fusion protein containing the anti-CD300c monoclonal antibody and IL-7 was removed by washing three times with 200 µL of PBST. 100 µL of 4 μg / mL detection antibody (HRP-α-human Fc specific IgG) diluted 1:1000 in blocking buffer was added, and the reaction was carried out at room temperature for 1 hour. After the reaction, the unbound detection antibody was removed by washing three times with 200 µL of PBST. Subsequently, TMB and hydrogen peroxide were mixed in a 1:1 ratio, 100 µL was added to each well, and the reaction was carried out at room temperature for 7 to 9 minutes. Afterward, 50 µL of 1 N sulfuric acid was added to stop the color development, and the binding affinity results were obtained by measuring at 450 nm using a microplate reader (product name: Varioskan LUX). The results are shown in Table 38 and Figure 33.

[0359]

[0360] Concentration (ug / ml) OD 3.5 3.4 16 3.2 76 0.6 25 3.3 54 2.9 08 0.1 56 3.0 28 2.2 32 0.0 39 1.6 43 0.6 53 0.0 10.5 04 0.2 7 0.0 20.1 52 0.0 87

[0361] As shown in Table 38 above, when the OD values ​​of the anti-CD300c monoclonal antibody (CL7) and the fusion protein (fusion protein CB701) containing the anti-CD300c monoclonal antibody and IL-7 were measured, the CB701 fusion protein maintained its binding ability to CD300c equivalent to that of the anti-CD300c monoclonal antibody despite structural modifications due to protein fusion, while it did not exhibit binding ability to CD300a. These results suggest that the intrinsic structure is stably maintained by binding via the G4S Linker, allowing for the expression of activity without affecting binding affinity. Furthermore, as shown in Figure 33, the sigmoid curve resulting from the binding ELISA also confirmed that the fusion protein of the present invention maintains the strong binding affinity of the anti-CD300c monoclonal antibody and binds to the CD300c antigen, thereby inducing continuous signal transduction and M1 macrophage differentiation. CD300c is an immunomodulatory receptor present on the surface of monocytes and macrophages, and when it binds to a ligand, it mediates immune-activating signaling to induce inflammation regulation and anti-tumor immune responses, which strongly suggests the effective tumor suppression and anticancer activity of the fusion protein of the present invention and supports the fact that the CB701 fusion protein is a promising therapeutic candidate capable of exhibiting anticancer effects through the regulation of the tumor microenvironment.

[0362]

[0363] Example 7.2. Confirmation of IL-7 receptor IL-7Rα binding ability of fusion protein CB701 (ELISA)

[0364] IL-7Rα (10758-IR, R&D systems), an IL-7 receptor, was dispensed into ELISA plates in coating buffer solution (0.1M sodium carbonate, pH 9.0) at a concentration of 4.0 µg / ml per well, and the plates were incubated at room temperature for 3 hours to bind CCR7 to the plates. Only PBS was dispensed into the blanks. Subsequently, unbound antigens were thoroughly removed by washing three times with PBST, after which 300 μL of PBST containing 5% BSA (bovine serum albumin) was added to each well and incubated at room temperature for 1 hour, followed by washing again with PBST.

[0365] Subsequently, the anti-CD300c monoclonal antibody and the fusion protein containing IL-7 were added in four-fold stepwise dilutions starting from a concentration of 2.5 µg / ml and reacted at room temperature for 1 hour to bind to the antigen. After 1 hour, the unbound fusion protein CB701 was removed by washing three times with PBST, and then a detection antibody (HRP-goat α-hIgG Fab, A0293, Sigma Aldrich) was added at a concentration of 1:500 and reacted again at room temperature for 1 hour. Afterward, the unbound detection antibody was removed with PBST, TMB solution was added and reacted for 8 minutes to develop color, after which 2 N sulfuric acid solution was added to terminate the color reaction, and the absorbance was measured at 450 nm to determine the specific binding ability of the anti-CD300c monoclonal antibody and the fusion protein containing IL-7 against IL-7Rα. The results are shown in Table 39 and Figure 34 below.

[0366]

[0367] EC50 of anti-CD300c monoclonal antibody and fusion protein containing IL-7 Dissociation constant (KD) of reference material IL7-Rα coating (1.0 µg / ml) IL7-Rα coating (2.0 µg / ml) 3.674 × 10⁻⁶9 M3.592 × 10 9 M3.1 × 10 7 M

[0368] As a result of measuring the Optical Density (OD) value of the fusion protein, there was no change in binding affinity for the receptor IL-7Rα. This result indicates that the original structure is stably maintained by binding via the G4S Linker, thereby exhibiting binding affinity without affecting the binding strength. Furthermore, the sigmoid curve resulting from the binding ELISA also confirmed that the fusion protein of the present invention binds to IL-7Rα, the receptor for IL-7, with strong binding strength.

[0369] In addition, it was shown that the fusion protein CB701 can bind to CD300c to promote the differentiation of monocytes into macrophages, thereby enhancing immune activity, while simultaneously binding to IL-7Rα, the receptor for IL-7, to induce T cell activation. Therefore, it was confirmed that the fusion protein CB701 exerts a synergistic anticancer effect by simultaneously inducing immune activation through CD300c regulation and increased IL-7Rα-mediated T cell activation, thereby strengthening the anticancer response of tumor-associated macrophages and T cells.

[0370]

[0371] Example 8. Confirmation of Cell Antigen Recognition by Fusion Protein CB701

[0372] To confirm that the fusion protein CB701 recognizes cell antigens, an experiment was designed to measure the binding of CD300c to the fusion protein CB701 using FACS after overexpressing CD300c in 293T cells (ATCC) and THP-1 cells (ATCC).

[0373] The fusion protein CB701 prepared in Example 6 will bind to CD300c overexpressed on the surface of THP-1 and 293T cells with strong binding affinity based on the S-shaped curve resulting from FACS binding.

[0374]

[0375] Example 9. Immune cell activation by fusion protein CB701

[0376] Example 9.1. Confirmation of T cell activation effect

[0377] In order to determine whether the fusion protein CB701 prepared in Example 6 can exhibit an anticancer effect through T cell activation, an experiment was designed to determine the production of IL-2 (Interleukin-2) in human T cells following treatment with the fusion protein CB701. IL-2 is an immune factor that helps T cells grow, proliferate, and differentiate. The purpose was to confirm that an increase in the production of IL-2 activates T cells by increasing the stimulation that induces increased differentiation, proliferation, and growth of T cells.

[0378] When Jurkat T cells activated by treatment with anti-CD3 monoclonal antibodies and anti-CD28 monoclonal antibodies are treated with the fusion protein CB701, the production of IL-2 increases compared to treatment with the anti-CD300c monoclonal antibody, and the fusion protein CB701 may be able to activate T cells more than the anti-CD300c monoclonal antibody, thereby inducing an anti-cancer immune response and inhibiting the growth of cancer tissue.

[0379]

[0380] Example 9.2. Confirmation of Promotion of Differentiation into M1 Macrophage (I): Measurement of Macrophage Differentiation Marker (TNF-α) Production

[0381] We designed an experiment to determine whether the fusion protein CB701 prepared in Example 6 can further promote the differentiation of mononuclear cells into M1 macrophages compared to the anti-CD300c monoclonal antibody by measuring the production of TNF-α (Tumor necrosis factor-α), a differentiation marker for M1 macrophages, using an ELISA kit (Human TNF-α Quantikine kit, R&D Systems), and also to confirm through experimental results using mouse macrophages (Raw264.7) whether the fusion protein CB701 can further promote the differentiation ability of mouse macrophages into M1 macrophages compared to the anti-CD300c monoclonal antibody.

[0382]

[0383] Experimental Example 9.3. Confirmation of Promotion of Differentiation Ability into M1 Macrophage (II): Observation of Cell Morphology

[0384] In order to confirm the differentiation pattern into M1 macrophages when the fusion protein CB701 prepared in Example 6 was treated to monocyte cells, 10 μg / ml of the fusion protein CB701 was treated to THP-1 cells, and after culturing for 48 hours, the cell morphology was observed under a microscope.

[0385] When treated with the fusion protein CB701, more THP-1 cells will change from suspension cells to round adherent cells, which are M1 macrophages, suggesting that treatment with the fusion protein CB701 further promotes the differentiation of monocyte cells into M1 macrophages.

[0386]

[0387] Example 9.4. Confirmation of Redifferentiation Ability of M2 Macrophages into M1 Macrophages (II): FACS Analysis

[0388] In order to determine whether the fusion protein CB701 prepared in Example 6 promotes differentiation into M1 macrophages when treated to monocyte cells, 10 μg / ml of the fusion protein CB701 was treated to THP-1 cells, and after culturing for 48 hours, markers expressed on the cell surface were stained and analyzed via FACS.

[0389] Compared to the control group, the experimental group treated with the fusion protein CB701 showed increased surface expression of the M0 macrophage marker and the M1 macrophage marker CD80, while decreasing the M2 marker CD206, suggesting that treatment with the fusion protein CB701 further promotes the differentiation of monocyte cells into M1 macrophages.

[0390]

[0391] Example 9.5. Confirmation of the redifferentiation ability of M2 macrophages into M1 macrophages

[0392] An experiment was designed to confirm whether the fusion protein CB701 prepared in Example 6 could further promote the differentiation of monocyte cells into M1 macrophages compared to the anti-CD300c monoclonal antibody by measuring the production of M1 macrophage differentiation markers TNF-α (Tumor necrosis factor-α), IL-1b, and IL-8 using an ELISA kit (R&D Systems).

[0393] In relation to the measurement of increased production of TNF-α, IL-1β, and IL-8, the fusion protein CB701 may be able to redifferentiate more M2 macrophages into M1 macrophages compared to the anti-CD300c monoclonal antibody.

[0394]

[0395] Experimental Example 9.6. Confirmation of increased migration of immune cells

[0396] In order to confirm the recruitment pattern of T cells caused by the fusion protein CB701 prepared in Example 6, an experiment was designed to observe the cells after treating wells with 200 nM of the fusion protein CB701, seeding Jurkat on a chamber, and culturing for 4 hours and 30 minutes.

[0397] As a result, administration of the fusion protein CB701 will increase the migration of Juckat cells compared to the control group, and thus the fusion protein CB701 will increase T cell recruitment more than the anti-CD300c monoclonal antibody and IL-7.

[0398]

[0399] Example 10. Confirmation of anticancer efficacy of fusion protein CB701

[0400] Example 10.1. Confirmation of human cancer cell growth inhibitory effect

[0401] We designed an experiment to determine the effect of the CD300c-targeting fusion protein CB701 on cancer cell growth compared to an anti-CD300c monoclonal antibody through cell proliferation analysis using A549 (human lung cancer cell line) and MDA-MB-231 (human breast cancer cell line). As a result, CB701 is expected to show superior cancer cell growth inhibitory effects compared to the anti-CD300c monoclonal antibody.

[0402]

[0403] Example 10.2. Confirmation of Inhibitory Effect on Mouse Cancer Cell Growth 1: Breast Cancer Cell Line

[0404] To confirm the breast cancer inhibitory effect of the fusion protein CB701 prepared in Example 6, an experiment was designed to establish a breast cancer orthotopic mouse model by injecting 4T1 (mouse breast cancer cell line) and to verify the anticancer effect through cell proliferation analysis. 1 × 10⁶ 4T1 breast cancer cells were injected into 7-week-old BALB / c mice. 5Prepare cells / mouse, mix Matrigel (Corning) and media in a 1:1 ratio, and inject 100 μL into the fourth teat of the mouse using a 1 mL syringe. Intravenous injection was selected as the route of administration for the fusion protein CB701; after disinfecting with a 70% alcohol swab, the injection was administered using a 1 mL syringe and a 30 G needle. The volume of the test substance excipient per mouse was set to 50 μL. Starting from day 5 after injection, the tumor volume within the mouse group was measured daily using a digital caliper to determine if it reached 50-100 mm³, and starting from day 7, the fusion protein CB701 was administered twice a week for a total of four doses.

[0405] As a result, while anti-CD300c monoclonal antibodies also have anticancer efficacy, the fusion protein CB701 will inhibit breast cancer cell growth even more significantly.

[0406]

[0407] Example 10.3. Confirmation of Inhibitory Effect on Mouse Cancer Cell Growth 2: Colorectal Cancer Cell Line

[0408] To confirm the colorectal cancer inhibitory effect of the fusion protein CB701 prepared in Example 6, an experiment was designed to establish a breast cancer orthotopic mouse model by injecting Colon26-Luc (mouse colorectal cancer cell line) and to confirm the anticancer effect through cell proliferation analysis.

[0409] 1×10 colon26 colorectal cancer cell line in 7-week-old BALB / c mice 6The cells / mouse mixture was prepared by mixing Matrigel (Corning) and media in a 1:1 ratio, and then injecting 50 μL of the mixture into the cecum of mice using a 30G needle. Intraperitoneal administration was selected as the route of administration for the fusion protein CB701; after disinfecting with a 70% alcohol swab, the injection was administered using an insulin syringe. The volume of the test substance excipient per animal was set at 50 μL. Colon26-Luc cells were injected into the mesentery of the cecum of BALB / c mice. Seven days after injection, the experimental animals were measured using a bioimaging (BLI) system and divided into five groups. CB701 was administered intraperitoneally twice a week for a total of four times.

[0410] As a result, the anti-CD300c monoclonal antibody is effective in treating colorectal cancer, and the fusion protein CB701 will also show an even greater effect in inhibiting the growth of colorectal cancer cells.

[0411]

[0412] Example 10.4. Confirmation of Inhibitory Effect on Mouse Cancer Cell Growth 3: Lung Cancer Cell Line

[0413] An experiment was designed to establish a non-small cell lung cancer orthotopic mouse model by injecting LLC-Luc (mouse non-small cell lung cancer cell line) using the fusion protein CB701 prepared in Example 6, and to confirm the anticancer effect through cell proliferation analysis.

[0414] 2×10 LLC-Luc lung cancer cell lines were injected into 7-week-old C57BL6J mice 4Prepared as cells / animal, Matrigel (Corning) and media were mixed in a 6:4 ratio, and a volume of 30 μL was injected into the left lung tissue of mice using a 30G needle. Intraperitoneal administration was selected as the route of administration for the fusion protein CB701; after disinfecting with a 70% alcohol swab, it was injected using an insulin syringe. The volume of the test substance excipient per animal was set at 50 μL. LLC-Luc cells were injected into the mice, and the fusion protein CB701 was administered twice a week for a total of four times, starting three days later.

[0415] As a result, the fusion protein CB701 will inhibit lung cancer cell growth more significantly than the anti-CD300c monoclonal antibody.

[0416]

[0417] Example 10.5. Confirmation of In vivo Anticancer Effect of Fusion Protein CB701

[0418] We administered the fusion protein CB701 or anti-CD300c monoclonal antibody to mice to observe an increase in M1-type tumor-associated macrophages and tumor-associated macrophages (TAM) in mouse cancer tissues.

[0419] We wanted to identify increased M1 form tumor-associated macrophages and tumor-associated macrophages (TAMs) in mouse cancer tissues due to CB701 treatment compared to treatment with anti-CD300c monoclonal antibody.

[0420] In addition, treatment with the fusion protein CB701 will increase the number of cytotoxic T cells and the activity of cytotoxic T cells more significantly compared to treatment with the anti-CD300c monoclonal antibody.

[0421] Based on the above results, the fusion protein CB701 will exhibit increased anticancer efficacy compared to the anti-CD300c monoclonal antibody under in vivo conditions.

[0422]

[0423] Example 11. Changes in biomarker expression following administration of fusion protein CB701

[0424] Example 11.1. Changes in the expression of immune cell-related markers and tumor microenvironment-related markers following administration of fusion protein CB701

[0425] To confirm changes in the expression of immune cell and tumor microenvironment-related markers when the fusion protein CB701 prepared in Example 6 was administered to a solid tumor model compared to the administration of an anti-CD300c monoclonal antibody, 2x10 colorectal cancer cell lines (CT26) 5 We designed an experiment to create an allogeneic mouse tumor model by transplanting a dog into an 8-week-old BALB / c mouse via subcutaneous injection.

[0426] The anti-CD300c monoclonal antibody or the fusion protein CB701 is administered to the above mouse tumor model, and changes in dendritic cell markers, macrophage markers, tumor microenvironment markers, Th1 response markers, or Th2 response markers following administration are observed.

[0427] When the fusion protein CB701 is administered, compared to the administration of the anti-CD300c monoclonal antibody, the expression of dendritic cell markers Bst2, CCL8, and Xcl1 will be significantly increased; the expression of M1 macrophage markers CCR7 and CD80 will be significantly increased; the expression of vegfa, pdgfrb, Col4a1, and Hif1a, which promote cancer growth in the tumor microenvironment, will be further decreased; and the expression of Th1 response markers Tbx21, Stat1, Stat4, Ifn-g, and Cxcr3 will be further increased.

[0428]

[0429] Example 11.2. Changes in the expression of immune checkpoint markers

[0430] We designed an experiment to determine which immune checkpoint markers showed a significant difference in expression compared to the control group when the fusion protein CB701 or the anti-CD300c monoclonal antibody was administered to an allogeneic mouse tumor model.

[0431] As a result, compared to the administration of the anti-CD300c monoclonal antibody, when the fusion protein CB701 is administered, the expression of inhibitory immune checkpoints PD-1, CTLA-4, and Lag3 will be increased, and the expression of agonistic immune checkpoints ICOS, OX40, Gitr, Cd27, and Cd28 will also be increased.

[0432]

[0433] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

Claims

Anti-CD300c antibody or its antigen-binding fragment; and a fusion protein comprising IL-7. Anti-CD300c antibody or its antigen-binding fragment; and a fusion protein comprising IL-7. A fusion protein according to claim 1, wherein the anti-CD300c antibody or its antigen-binding fragment comprises heavy chain CDR1 of SEQ ID NO. 3; heavy chain CDR2 of SEQ ID NO. 5; heavy chain CDR3 of SEQ ID NO. 7; light chain CDR4 of SEQ ID NO. 11; light chain CDR5 of SEQ ID NO. 13; and light chain CDR6 of SEQ ID NO.

15. A fusion protein according to claim 1, wherein the anti-CD300c antibody or the antigen-binding fragment thereof comprises heavy chain FR1 of SEQ ID NO. 17; heavy chain FR2 of SEQ ID NO. 19; heavy chain FR3 of SEQ ID NO. 21; heavy chain FR4 of SEQ ID NO. 23; light chain FR5 of SEQ ID NO. 25; light chain FR6 of SEQ ID NO. 27; light chain FR7 of SEQ ID NO. 29; and light chain FR8 of SEQ ID NO.

31. A fusion protein according to claim 1, wherein the anti-CD300c antibody or its antigen-binding fragment comprises the heavy chain variable region of SEQ ID NO. 1 and the light chain variable region of SEQ ID NO.

9. A fusion protein according to claim 1, wherein the CD300c antibody or its antigen-binding fragment regulates the expression level of one or more genes selected from the factors listed in Tables 4 to 36. A fusion protein according to claim 1, wherein the IL-7 comprises the amino acid sequence of SEQ ID NO. 33 or 35. The fusion protein of claim 1, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO. 37, 39, or 40. In claim 1, the anti-CD300c antibody or its antigen-binding fragment; and a fusion protein in which IL-7 is linked by a linker. The fusion protein according to claim 1, wherein the fusion protein simultaneously performs anticancer immune activation through CD300c response regulation and T cell activity enhancement. The fusion protein of claim 1, wherein the fusion protein enhances a T-cell-based anticancer immune response by restoring IL-7 levels reduced by anti-CD300c antibody treatment. A polynucleotide encoding a fusion protein of any one of claims 1 to 10. A vector comprising a polynucleotide encoding a fusion protein of any one of claims 1 to 10. A host cell comprising one or more of the following: a fusion protein of any one of claims 1 to 10; a polynucleotide encoding the same; and a vector containing said polynucleotide. A pharmaceutical composition for the prevention or treatment of cancer, comprising a fusion protein of any one of claims 1 to 10. A pharmaceutical composition according to claim 14, wherein the cancer comprises one or more selected from the group consisting of pancreatic cancer, gastric cancer, colorectal cancer, bile duct cancer, esophageal cancer, rectal cancer, oral cancer, pharyngeal cancer, laryngeal cancer, lung cancer, colon cancer, breast cancer, ovarian cancer, cervical cancer, endometrial cancer, prostate cancer, testicular cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, bone cancer, connective tissue cancer, skin cancer, tongue cancer, brain cancer, thyroid cancer, leukemia, Hodgkin's disease, lymphoma, and blood cancer. A pharmaceutical composition according to claim 14, wherein the cancer comprises one or more selected from the group consisting of colorectal cancer, blood cancer, breast cancer, and lung cancer. A food composition for preventing or improving cancer, comprising a fusion protein of any one of claims 1 to 10. A method for preventing or treating cancer, comprising the step of administering a fusion protein of any one of claims 1 to 10 to an individual. A method for producing a fusion protein comprising the step of culturing a host cell in a medium comprising: a fusion protein of any one of claims 1 to 10; a polynucleotide encoding the same; and one or more vectors comprising said polynucleotide.

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