Anti-CD300c monoclonal antibody and its biomarker for cancer prevention or treatment

Abstract

The present invention relates to an anti-CD300c monoclonal antibody and its use for preventing or treating cancer. The anti-CD300c monoclonal antibody according to the present invention is expected to be effectively used for the growth, development, metastasis, etc. of various cancers by not only binding to the CD300c antigen with high specificity but also promoting anti-cancer immunity.

Description

[Technical Field]

[0001] The present invention relates to an anti-CD300c antibody or an antigen-binding fragment thereof, as well as a biomarker, composition, method and kit for preventing or treating cancer, comprising the same. [Background technology]

[0002] Cancer, one of the leading causes of death in modern society, is a disease that develops when normal cells change due to genetic mutations caused by various factors. It is a malignant form of tumor that does not follow the differentiation, proliferation, or growth morphology of normal cells. Cancer is characterized by uncontrolled cell growth. This abnormal cell growth forms a cell mass called a tumor, which infiltrates surrounding tissues and, in severe cases, can metastasize to other organs in the body. Cancer is an intractable chronic disease that often fails to cure fundamental disease, even with treatments such as surgery, radiation, and chemotherapy, causing pain and ultimately leading to death. In particular, the global cancer incidence rate has been increasing by more than 5% annually due to factors such as an increasing aging population and environmental degradation. According to a WHO report, the number of people diagnosed with cancer is estimated to increase to 30 million within the next 25 years, of which 20 million will die from cancer.

[0003] Cancer drug treatments, i.e., anticancer drugs, generally treat cancer by attacking and killing cancer cells as cytotoxic compounds. However, they cause significant side effects because they damage not only cancer cells but also normal cells. Therefore, targeted anticancer drugs have been developed to reduce side effects. However, while these targeted anticancer drugs have reduced side effects, they have a limiting effect: a high rate of resistance. Therefore, in recent years, interest has been growing in immune-mediated anticancer drugs that utilize the body's immune system to reduce problems associated with toxicity and resistance. One example of such immune-mediated anticancer drugs is an immune barrier inhibitor, which specifically binds to PD-L1 on the surface of cancer cells and inhibits T cell binding to PD-1, thereby activating T cells to attack cancer cells. However, these immune barrier inhibitors are not effective in a wide variety of cancers, and there is a pressing need for the development of new anti-inflammatory immune therapeutic agents that are equally effective across a wide range of cancers. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Korean Patent Publication No. 10-2018-0099557 Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to solve all of the above problems.

[0006] An object of the present invention is to provide an anti-CD300c antibody for the prevention or treatment of cancer.

[0007] Another object of the present invention is to provide an anti-cancer therapy using an anti-CD300c antibody.

[0008] Another object of the present invention is to provide a pharmaceutical composition for therapy using an anti-CD300c antibody for the prevention or treatment of cancer.

[0009] Another object of the present invention is to provide a method for preventing or treating cancer using an anti-CD300c antibody.

[0010] Another object of the present invention is to provide a kit for therapy using an anti-CD300c antibody for the prevention or treatment of cancer.

[0011] The objects of the present invention are not limited to the above-mentioned objects, but will become more apparent from the following description and can be realized by the means and combinations thereof set forth in the claims. [Means for solving the problem]

[0012] A typical configuration of the present invention to achieve the above object is as follows.

[0013] According to one aspect of the present invention, there is provided an antibody (eg, a monoclonal antibody) or an antigen-binding fragment thereof that specifically binds to CD300c.

[0014] Another aspect of the present invention provides an anti-CD300c antibody or an antigen-binding fragment thereof and use thereof for the prevention or treatment of cancer.

[0015] Another aspect of the present invention provides use of an anti-CD300c antibody or an antigen-binding fragment thereof for the manufacture of a medicament for the prevention or treatment of cancer.

[0016] Another aspect of the present invention provides an anti-cancer therapy comprising an anti-CD300c antibody or an antigen-binding fragment thereof as an active ingredient.

[0017] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising an anti-CD300c antibody or an antigen-binding fragment thereof as an active ingredient.

[0018] Another aspect of the present invention provides a method for preventing or treating cancer, comprising administering an anti-CD300c antibody or an antigen-binding fragment thereof to a subject in need thereof.

[0019] According to another aspect of the present invention, there is provided a kit for preventing or treating cancer, comprising a composition containing an effective amount of an anti-CD300c antibody or its antigen-binding fragment, and instructions for using the antibody or its antigen-binding fragment. [Effects of the Invention]

[0020] The anti-CD300c monoclonal antibody of the present invention specifically binds with high avidity to CD300c expressed on the surface of various cancers, thereby activating T cells and promoting differentiation into M1 macrophages, thereby effectively inhibiting cancer cell proliferation and making it an effective immunotherapeutic agent for various cancers. Furthermore, the anti-CD300c monoclonal antibody of the present invention not only further enhances the therapeutic effect when administered in combination with existing immunotherapeutic anticancer drugs, but also has cross-reactivity, making it applicable to a wide range of mammals. Furthermore, when treated with the anti-CD300c monoclonal antibody of the present invention on resistant cancer cells that exhibit the ability to resist apoptosis, it is expected to significantly weaken the resistance of the cancer cells and exhibit excellent efficacy in preventing cancer recurrence. In addition, cancer cells generally evade the immune system by inhibiting the production of IL-2, a pro-inflammatory cytokine. Anti-CD300c monoclonal antibodies have been shown to restore the production of IL-2 blocked by cancer cells, thereby inducing cancer cell death through an activated immune system, and are expected to be used as a more fundamental immune anti-cancer agent. [Brief explanation of the drawings]

[0021] [Figures 1a-1y]The heavy and light chain variable region sequences (nucleic acid and amino acid sequences) of each of the 25 anti-CD300c monoclonal antibodies according to the present invention are shown. In each figure, the CDR regions (CDR1, CDR2, and CDR3) are indicated in order. [Figure 2] FIG. 1 is a simplified schematic diagram illustrating the mechanism by which the anti-CD300c monoclonal antibody and / or CD300c siRNA of the present invention exerts its anti-cancer effect. [Figure 3] FIG. 1 is a simplified schematic diagram showing the mechanisms by which the anti-CD300c monoclonal antibodies of the present invention act on monocytes, T cells, and cancer cells, respectively. [Figure 4] 1 shows the results of SDS-PAGE under non-reducing conditions of the anti-CD300c monoclonal antibody according to Example 1.4. [Figure 5] 1 shows the results of SDS-PAGE under reducing conditions of the anti-CD300c monoclonal antibody according to Example 1.4. [Figure 6] 1 shows the results of comparing CD300c expression in normal cells, immune cells, and cancer cell lines according to Experimental Example 1.1. [Figure 7a] 1 shows the results of confirming the expression of CD300c in cancer tissues in Experimental Example 1.2. [Figure 7b] 1 shows the results of confirming the expression of CD300c in immune cells in Experimental Example 1.2. [Figure 8a] 1 shows the results of confirming the expression of CD300c in tonsillar tissue in Experimental Example 1.3. [Figure 8b] 1 shows the results of confirming the expression of CD300c in cancer tissues in Experimental Example 1.3. [Figure 9] 1 shows the results of confirming the binding ability of anti-CD300c monoclonal antibodies to the CD300c antigen in Experimental Example 2.1. [Figure 10] 1 shows a sigmoidal curve resulting from FACS binding in Experimental Example 2.2. [Figure 11] 1 shows the results of binding ELISA according to Experimental Example 2.3. [Figure 12] 1 shows the results of surface plasmon resonance in Experimental Example 2.4. [Figure 13] 1 shows the results of binding ELISA according to Experimental Example 2.5. [Figure 14] 1 shows the results of binding ELISA according to Experimental Example 2.6. [Figure 15] In relation to Experimental Example 2.7, the results of a comparison of overall survival time in patients with various cancers according to CD300c expression level are shown. [Figure 16] 3 shows the results of Experimental Example 3.1 confirming the anti-cancer effect of anti-CD300c monoclonal antibody through T cell activation. [Figure 17-18] 1 shows the results of confirming the effect of anti-CD300c monoclonal antibody on differentiation into M1 macrophages in Experimental Example 3.2. [Figure 19-20] 1 shows the results of confirming the concentration-dependent effect of anti-CD300c monoclonal antibody on differentiation into M1 macrophages in Experimental Example 3.3. [Figure 21] 1 shows the results of confirming the effect of anti-CD300c monoclonal antibody on differentiation into M1 macrophages in Experimental Example 3.4. [Figure 22] 1 shows the results of reconfirming whether anti-CD300c monoclonal antibody promotes differentiation of human monocytes into M1 macrophages in Experimental Example 3.5. [Figure 23-25] 1 shows the results of confirming whether anti-CD300c monoclonal antibody in Experimental Example 3.6 redifferentiates M2 macrophages into M1 macrophages. [Figure 26] 1 shows the results of confirming the differentiation and redifferentiation abilities of anti-CD300c monoclonal antibodies into M1 macrophages in Experimental Example 3.7. [Figure 27] 4 shows the results of Experimental Example 4.1, which confirmed the effect of a monoclonal antibody targeting CD300c on the growth of cancer cells. [Figure 28] 4 shows the results of Experimental Example 4.1, which confirmed the effect of a monoclonal antibody targeting CD300c on the growth of cancer cells. [Figure 29] 4 shows the results of confirming the inhibitory effect of anti-CD300c monoclonal antibody on cancer cell growth depending on the concentration in Experimental Example 4.2. [Figure 30] 4 shows the results of confirming whether anti-CD300c monoclonal antibody promotes the differentiation of mouse macrophages into M1 macrophages in Experimental Example 4.3. [Figure 31] 4 shows the results of confirming whether the anti-CD300c monoclonal antibody of Experimental Example 4.4 has an anti-cancer effect. [Figure 32] 5 shows the results of a comparison of the M1 macrophage differentiation ability of anti-CD300c monoclonal antibody and existing immunological anticancer agents in Experimental Example 5.1. [Figure 33] 5 shows the results of a comparison of the M1 macrophage differentiation ability of anti-CD300c monoclonal antibody and existing immunological anticancer agents in Experimental Example 5.1. [Figure 34] 5 shows the results of a comparison of the M1 macrophage differentiation ability of anti-CD300c monoclonal antibody and existing immunological anticancer agents in Experimental Example 5.1. [Figure 35] 5 shows the results of a comparison of the M1 macrophage differentiation ability of anti-CD300c monoclonal antibody and existing immunological anticancer agents in Experimental Example 5.1. [Figure 36] 5 shows the results of a comparison of the differentiation ability of M0 macrophages into M1 macrophages between anti-CD300c monoclonal antibody and existing immunological anticancer agents in Experimental Example 5.2. [Figure 37] 5 shows the results of a comparison of the differentiation ability into M1 macrophages between anti-CD300c monoclonal antibody and existing immunological anti-cancer agents in Experimental Example 5.3. [Figure 38] Experimental Example 5.4 shows the results of a comparison of the cancer cell growth inhibitory effects between anti-CD300c monoclonal antibody and existing immunological anticancer agents. [Figure 39] Experimental Example 5.4 shows the results of a comparison of the cancer cell growth inhibitory effects between anti-CD300c monoclonal antibody and existing immunological anticancer agents. [Figure 40] 6 shows the results of in vivo confirmation of the effect of anti-CD300c monoclonal antibody on tumor-associated macrophages according to Experimental Example 6.1. [Figure 41]6 shows the results of in vivo testing of the effect of anti-CD300c monoclonal antibody on CD8+ T cells according to Experimental Example 6.2. [Figure 42] 6 shows the results of confirming whether anti-CD300c monoclonal antibody increases the number of CD8+ T cells in a tumor-specific manner according to Experimental Example 6.3. [Figure 43] 6 shows the results of in vivo testing of the effect of anti-CD300c monoclonal antibody on increasing CD8+ T cell activity according to Experimental Example 6.4. [Figure 44] 6 shows the results of in vivo testing of the effect of anti-CD300c monoclonal antibody on increasing cytotoxic T cells compared to regulatory T cells according to Experimental Example 6.5. [Figure 45] Experimental Example 6.6 shows the results of confirming the effects of anti-CD300c monoclonal antibodies on cytotoxic T cells, regulatory T cells, and tumor-associated macrophages. [Figure 46] 6 shows the results of confirming the anti-cancer effect of anti-CD300c monoclonal antibody under in vivo conditions in Experimental Example 6.7. [Figure 47] 2 shows the results of Nanostring immune profiling obtained when a solid tumor model was treated with CL7 according to Example 2.1. [Figure 48] This figure shows the changes in expression of various immune cell- and tumor microenvironment-related markers obtained when CL7 was treated in a solid tumor model according to Example 2.1. * indicates markers whose expression levels were statistically significantly changed compared to before CL7 treatment. [Figure 49] Example 2.2 shows changes in the expression of immune barrier markers confirmed based on the NanoString immune profiling results obtained in Example 2.1. * indicates markers whose expression levels were statistically significantly changed compared to before CL7 treatment. [Figure 50] 7 shows the results of differentiation of monocytes into M1 macrophages (presence or absence of increase in M1 macrophages) by treatment with anti-CD300c monoclonal antibody and an immunosuppressant, alone or in combination, according to Experimental Example 7.1. [Figure 51] 1 shows the results of differentiation of monocytes into M1 macrophages by treatment with anti-CD300c monoclonal antibody in Experimental Example 7.2 (showing whether or not there is an increase in M1 macrophage markers). [Figure 52] 7 shows the results of differentiation of monocytes into M1 macrophages (indicating whether or not there is an increase in M1 macrophage markers) by combined treatment with anti-CD300c monoclonal antibody and an immunosuppressant in Experimental Example 7.2. [Figure 53] 7 shows the results of confirming MAPK signal transduction, which is a signal for M1 macrophage differentiation, during combined treatment with anti-CD300c monoclonal antibody and an immunosuppressant in Experimental Example 7.4. [Figure 54] 7 shows the results of confirming the signal transduction of NF-kB, which is a signal for M1 macrophage differentiation, during combined treatment with anti-CD300c monoclonal antibody and an immunosuppressant in Experimental Example 7.4. [Figure 55] 7 shows the results of confirming the signal transduction of IkB, which is a signal for M1 macrophage differentiation, during combined treatment with anti-CD300c monoclonal antibody and an immunosuppressant in Experimental Example 7.4. [Figure 56] 8 shows the results of confirming changes in apoptosis signals during combined treatment with anti-CD300c monoclonal antibody and immunosuppressant in Experimental Example 8.1. [Figure 57] 8 shows the results of confirming the growth inhibitory effect of cancer cells when anti-CD300c monoclonal antibody and immunosuppressant were treated in combination in Experimental Example 8.2. [Figure 58] 8 shows the results of confirming the growth inhibitory effect of cancer cells when anti-CD300c monoclonal antibody and immunosuppressant were treated in combination in Experimental Example 8.2. [Figure 59] The experimental method used in Experimental Example 9.1 is shown in outline. [Figure 60] 9 shows the in vivo tumor growth inhibitory effect observed when anti-PD-1 antibody and anti-CD300c monoclonal antibody according to Experimental Example 9.1 were administered alone or in combination to mice transplanted with colon cancer cell lines. [Figure 61]This shows the results of confirming whether anti-CD300c monoclonal antibody increases M1 macrophages in cancer tissue in a mouse model in Experimental Example 9.3. [Figure 62] Experimental Example 9.4 shows the results of confirming whether anti-CD300c monoclonal antibody promotes CD8+ T cell immunity in a mouse tumor model. [Figure 63] The experimental method used in Experimental Example 10.1 is shown in outline. [Figure 64] The results of Experimental Example 10.1 confirming whether the anti-CD300c monoclonal antibody is effective in other carcinomas in addition to the CT26 colon cancer mouse model are shown below. [Figure 65] Experimental Example 10.2 shows the results of in vivo investigation of the effects of single or combined administration of anti-CD300c monoclonal antibody and immuno-anticancer agent (including double and triple combination administration) on CD8+ T cells in a B16F10 melanoma model. [Figure 66] Experimental Example 10.3 shows the effects of single or combined administration of anti-CD300c monoclonal antibody and immunosuppressant (including double and triple combination administration) on regulatory T cells in a B16F10 melanoma model under in vivo conditions. [Figure 67] Experimental Example 10.4 shows the results of in vivo examination of the effects of single or combined administration (including double and triple combined administration) of anti-CD300c monoclonal antibody and immunoanticancer agent on macrophages in a B16F10 melanoma model. [Figure 68a] 1 shows the results of confirming the anti-cancer effect under in vivo conditions by combined administration of an anti-CD300c monoclonal antibody and an immunological anti-cancer agent according to Experimental Example 11. The figures show the rate of reduction in tumor volume. [Figure 68b] 1 shows the results of in vivo confirmation of the anti-cancer effect of combined administration of an anti-CD300c monoclonal antibody and an immunological anti-cancer agent according to Experimental Example 11. The complete remission rate is shown. [Figure 69] 1 shows the results of Experimental Example 12 confirming the effect of combined administration of an anti-CD300c monoclonal antibody and an immunological anticancer agent in improving long-term survival rate. [Figure 70]1 shows the results of in vivo confirmation of the effect of combined administration of anti-CD300c monoclonal antibody and an immunological anticancer agent in Experimental Example 13 on preventing cancer recurrence. [Figure 71] 1 shows the results of Experimental Example 14 confirming the immunological memory effect caused by the combined administration of an anti-CD300c monoclonal antibody and an immunological anticancer agent. [Fig. 72a-72b] 15 shows the results of confirming whether single or combined administration (including double and triple combined administration) of anti-CD300c monoclonal antibody and immunoanticancer agent promotes differentiation of monocytes into M1 macrophages. [Figure 73a] 1 shows the results of confirming whether the anti-CD300c monoclonal antibody of Experimental Example 16 can suppress the growth of cancer cells when administered in combination with an immunosuppressive anticancer agent. [Figure 73b] 1 shows the results of confirming whether the anti-CD300c monoclonal antibody of Experimental Example 16 can suppress the growth of cancer cells when administered in combination with an immunosuppressive anticancer agent. DETAILED DESCRIPTION OF THE INVENTION

[0022] The following detailed description of the present invention will be described with reference to certain drawings and with reference to specific embodiments in which the present invention may be practiced; however, the present invention is not limited thereto, but rather only by the appended claims, along with the full scope of equivalents thereof, which, when properly interpreted, are to be claimed. It should be understood that the various embodiments of the present invention, although different from one another, are not necessarily mutually exclusive. For example, specific shapes, structures, and characteristics described herein may be modified from one embodiment to another, or may be embodied in combination, without departing from the spirit and scope of the present invention. Technical and scientific terms used herein have the same meanings as commonly used in the art to which the present invention pertains, unless otherwise defined. For purposes of interpreting this specification, the following definitions shall apply, and terms used in the singular shall include the plural where appropriate, and vice versa.

[0023] definition As used herein, the term "about" refers to a normal range of error for the respective value known to one of ordinary skill in the art.

[0024] The term "antibody" is used broadly and includes monoclonal antibodies (including full-length antibodies) of any isotype, such as IgG, IgM, IgA, IgD, and IgE, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), antibody fusions (e.g., antibody-(poly)peptide fusions or antibody-compound fusions), and antibody fragments (including antigen-binding fragments). As used herein, the prefix "anti-," when used in reference to an antigen, indicates that the antibody is reactive with that antigen. Antibodies reactive with a particular antigen can be generated by synthetic and / or recombinant methods, such as selection of recombinant antibody libraries with phage or similar vectors, or by immunization of animals with the antigen or antigen-encoding nucleic acid, but are not limited to these. A typical IgG antibody is composed of two identical heavy chains and two identical light chains linked by disulfide bonds. Each heavy and light chain contains a constant region and a variable region. The heavy chain variable region (HVR) and light chain variable region (LVR) each contain three segments called "complementarity determining regions" ("CDRs") or "hypervariable regions," which are primarily involved in binding to antigen epitopes. These are numbered sequentially from the N-terminus and are usually called CDR1, CDR2, and CDR3. The more highly conserved regions in the variable regions outside the CDRs are called "framework regions" ("FRs"). As used herein, antibodies may be, for example, animal antibodies, chimeric antibodies, humanized antibodies, or human antibodies.

[0025] The term "humanization" (also called reshaping or CDR grafting) includes established techniques for reducing the immunogenicity and improving the affinity or effector functions (ADCC, complement activation, Clq binding) of monoclonal antibodies derived from xenogeneic sources (usually rodents).

[0026] The term "monoclonal antibody" is used interchangeably with "monoclonal antibody" and refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation), which may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. A monoclonal antibody exhibits the characteristics of an antibody obtained from a substantially homogeneous population and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention can be produced by a variety of techniques, including hybridoma methods, recombinant DNA methods, phage display technology, and methods utilizing transgenic animals containing all or part of the human immunoglobulin locus.

[0027] The term "antigen-binding fragment" refers to a portion of an antibody or a polypeptide comprising the same that has the ability to specifically bind to an antigen. Unless "antibody" is specifically understood to exclude "antigen-binding fragment" depending on the context, "antibody" and "antigen-binding fragment" are used interchangeably, and "antibody" can be interpreted as including "antigen-binding fragment." Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, triabodies, tetrabodies, cross-Fab fragments, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed from antibody fragments and single-domain antibodies.

[0028] The term "anticancer agent" is a general term for known drugs used in existing cancer treatments that exert cytotoxic or cytostatic effects on cancer cells by acting on various metabolic pathways of cells, and includes chemical anticancer agents, targeted anticancer agents, and immunological anticancer agents.

[0029] The term "immunotherapy" refers to an agent that activates immune cells to kill cancer cells.

[0030] The term "subject" is used interchangeably with "patient" and may refer to a mammal in need of cancer prevention or treatment, such as a primate (e.g., human), pet animal (e.g., dog, cat, etc.), livestock animal (e.g., cow, pig, horse, sheep, goat, etc.), or laboratory animal (e.g., rat, mouse, guinea pig, etc.). In one embodiment of the present invention, the subject is a human.

[0031] The term "treatment" generally refers to obtaining a desired pharmacological and / or physiological effect. Such an effect has a therapeutic effect in that it partially or completely cures a disease and / or the deleterious effects of such a disease. Preferred therapeutic effects include, but are not limited to, preventing the onset or recurrence of a disease, ameliorating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, reducing the rate of disease progression, reversing or alleviating the disease state, and improving or improving the prognosis. Preferably, "treatment" may refer to medical intervention of an already existing disease or disorder.

[0032] The term "prevention" relates to prophylactic treatment, i.e., a measure or procedure intended to prevent rather than treat a disease. "Prevention" means obtaining a desired prophylactic pharmacological and / or physiological effect in terms of partially or completely preventing a disease or its symptoms.

[0033] The term "administration" means providing a substance (e.g., anti-CD300c antibodies and antigen-binding fragments thereof or other anti-cancer agents) to a subject to achieve a prophylactic or therapeutic purpose (e.g., cancer prevention or treatment).

[0034] The term "biological sample" encompasses a variety of sample types obtained from a subject and may be used in diagnostic or monitoring assays. Biological samples include, but are not limited to, blood and other liquid samples of biological origin, biopsy samples, tissue cultures, or solid tissue samples such as cells derived therefrom and their progeny. Thus, biological samples encompass clinical samples, cells in culture, cell supernatants, cell lysates, serum, plasma, biological fluids, and tissue samples, particularly tumor samples. The term "biological material" refers to any analytical material obtained using the biological sample.

[0035] The term "expression level" can be determined by measuring the expression level of one or more of the marker's mRNA and protein. Any method known in the art can be used to measure mRNA or protein expression levels. For example, an agent for measuring mRNA expression levels can be a primer pair or probe that specifically binds to the marker gene, and an agent for measuring protein expression levels can be an antibody, substrate, ligand, or cofactor that specifically binds to the marker. Analytical methods for measuring mRNA expression levels include, but are not limited to, reverse transcriptase polymerase reaction, competitive reverse transcriptase polymerase reaction, real-time reverse transcriptase polymerase reaction, RNase protection assay, Northern blotting, and DNA chips. Analytical methods for measuring protein levels include, but are not limited to, Western blot, ELISA, radioimmunoassay, radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation analysis, complement fixation analysis, FACS, and protein chips.

[0036] The term "therapeutic responsiveness" refers to whether an individual suffering from or suspected of suffering from cancer responds favorably or unfavorably to treatment using an active therapeutic ingredient (e.g., a CD300c antibody or its antigen-binding fragment), and can be assessed by changes in the immune system associated with tumor treatment that appear after administration of an anti-CD300c antibody or its antigen-binding fragment.

[0037] Anti-CD300c antibody One aspect of the present invention provides an anti-CD300c antibody or antigen-binding fragment thereof. The anti-CD300c antibody or antigen-binding fragment thereof according to the present invention is an antigen-binding molecule that specifically binds to CD300c protein. Preferably, the anti-CD300c antibody or antigen-binding fragment thereof is a monoclonal antibody or antigen-binding fragment thereof that specifically binds to CD300c protein.

[0038] The term "CD300c protein" is used interchangeably with "CD300c" or "CD300c antigen." As a protein encoded by the CD300c gene, it shows considerable sequence identity with B7 family proteins and is known to be expressed on the membrane of antigen-presenting cells. Suppression of CD300c protein expression or activity can induce T cell activation and / or promote differentiation into M1 macrophages.

[0039] The term "anti-CD300c antibody" can also be used interchangeably with "polypeptide" that binds to CD300c protein. The term "polypeptide" is intended to mean any polymer composed of amino acids linked together through peptide bonds, regardless of length. That is, as used herein, polypeptide includes peptides and proteins.

[0040] In one embodiment, the anti-CD300c antibody or antigen-binding fragment thereof may specifically bind to the extracellular domain (ECD) of the CD300c protein. The extracellular domain of CD300c may be the extracellular domain of human CD300c protein. The extracellular domain of CD300c may comprise the amino acid sequence set forth in SEQ ID NO: 402.

[0041] In one example, it was confirmed that the expression level of CD300c protein is highly correlated with the survival time of various cancer patients. Specifically, it was confirmed that cancer patients with a high CD300c expression level, relative to the average CD300c expression level, had a shorter survival time than cancer patients with a low CD300c expression level. This indicates that inhibition of CD300c expression or activity using the anti-CD300c antibody or antigen-binding fragment thereof according to the present invention is effective in treating cancer or prolonging the survival time of cancer patients.

[0042] The anti-CD300c antibody or antigen-binding fragment thereof according to the present invention can exert an anti-cancer effect by specifically binding to CD300c expressed on the surface of various cancer cells. The binding of the anti-CD300c antibody to CD300c activates T cells and promotes their differentiation into M1 macrophages, effectively suppressing cancer cell proliferation. This allows the anti-CD300c antibody to be effectively used as an immunotherapeutic agent for various cancers. Furthermore, the therapeutic effect of this anti-CD300c antibody can be further enhanced by coadministration with existing anti-cancer drugs. Furthermore, due to its species cross-reactivity (e.g., between human and mouse antigens), it can be widely used in various mammals. Furthermore, when this anti-CD300c antibody is administered to resistant cancer cells that exhibit the ability to resist apoptosis, it significantly weakens the resistance of the cancer cells, and is therefore expected to exhibit excellent efficacy in preventing cancer recurrence. Furthermore, cancer cells generally evade the immune system by inhibiting the production of IL-2, a pro-inflammatory cytokine. Anti-CD300c antibodies have been shown to induce cancer cell death through an activated immune system by restoring the production of IL-2 blocked by cancer cells. Therefore, they are expected to be used as a more fundamental immune-mediated anti-cancer agent. For details regarding CD300c proteins or anti-CD300c antibodies, please refer to Korean Patent Publication No. 10-2019-0136949, the contents of which are incorporated herein in their entirety.

[0043] In one embodiment, the anti-CD300c monoclonal antibody or antigen-binding fragment thereof is (i) a CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:19, SEQ ID NO:31, SEQ ID NO:43, SEQ ID NO:55, SEQ ID NO:67, SEQ ID NO:79, SEQ ID NO:91, SEQ ID NO:103, SEQ ID NO:115, SEQ ID NO:127, SEQ ID NO:139, SEQ ID NO:151, SEQ ID NO:163, SEQ ID NO:175, SEQ ID NO:187, SEQ ID NO:199, SEQ ID NO:211, SEQ ID NO:223, SEQ ID NO:235, SEQ ID NO:247, SEQ ID NO:259, SEQ ID NO:271, SEQ ID NO:283, and SEQ ID NO:295; a CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:20, SEQ ID NO:32, SEQ ID NO:44, SEQ ID NO:56, SEQ ID NO:68, SEQ ID NO:80, SEQ ID NO:92, SEQ ID NO:104, SEQ ID NO:116, SEQ ID NO:128, SEQ ID NO:140, SEQ ID NO:152, SEQ ID NO:164, SEQ ID NO:176, SEQ ID NO:188, SEQ ID NO:200, SEQ ID NO:212, SEQ ID NO:224, SEQ ID NO:236, SEQ ID NO:248, SEQ ID NO:260, SEQ ID NO:272, SEQ ID NO:284 and SEQ ID NO:296; and a heavy chain variable region comprising a CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO:33, SEQ ID NO:45, SEQ ID NO:57, SEQ ID NO:69, SEQ ID NO:81, SEQ ID NO:93, SEQ ID NO:105, SEQ ID NO:117, SEQ ID NO:129, SEQ ID NO:141, SEQ ID NO:153, SEQ ID NO:165, SEQ ID NO:177, SEQ ID NO:189, SEQ ID NO:201, SEQ ID NO:213, SEQ ID NO:225, SEQ ID NO:237, SEQ ID NO:249, SEQ ID NO:261, SEQ ID NO:273, SEQ ID NO:285, and SEQ ID NO:297; and (ii) a CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:22, SEQ ID NO:34, SEQ ID NO:46, SEQ ID NO:58, SEQ ID NO:70, SEQ ID NO:82, SEQ ID NO:94, SEQ ID NO:106, SEQ ID NO:118, SEQ ID NO:130, SEQ ID NO:142, SEQ ID NO:154, SEQ ID NO:166, SEQ ID NO:178, SEQ ID NO:190, SEQ ID NO:202, SEQ ID NO:214, SEQ ID NO:226, SEQ ID NO:238, SEQ ID NO:250, SEQ ID NO:262, SEQ ID NO:274, SEQ ID NO:286, and SEQ ID NO:298; a CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:23, SEQ ID NO:35, SEQ ID NO:47, SEQ ID NO:59, SEQ ID NO:71, SEQ ID NO:83, SEQ ID NO:95, SEQ ID NO:107, SEQ ID NO:119, SEQ ID NO:131, SEQ ID NO:143, SEQ ID NO:155, SEQ ID NO:167, SEQ ID NO:179, SEQ ID NO:191, SEQ ID NO:203, SEQ ID NO:215, SEQ ID NO:227, SEQ ID NO:239, SEQ ID NO:251, SEQ ID NO:263, SEQ ID NO:275, SEQ ID NO:287 and SEQ ID NO:299; and The light chain variable region may comprise a CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:24, SEQ ID NO:36, SEQ ID NO:48, SEQ ID NO:60, SEQ ID NO:72, SEQ ID NO:84, SEQ ID NO:96, SEQ ID NO:108, SEQ ID NO:120, SEQ ID NO:132, SEQ ID NO:144, SEQ ID NO:156, SEQ ID NO:168, SEQ ID NO:180, SEQ ID NO:192, SEQ ID NO:204, SEQ ID NO:216, SEQ ID NO:228, SEQ ID NO:240, SEQ ID NO:252, SEQ ID NO:264, SEQ ID NO:276, SEQ ID NO:288, and SEQ ID NO:300.

[0044] In other embodiments, the heavy chain variable region comprises: (i) a CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:19, SEQ ID NO:43, SEQ ID NO:55, SEQ ID NO:67, SEQ ID NO:79, SEQ ID NO:103, SEQ ID NO:115, SEQ ID NO:127, SEQ ID NO:139, SEQ ID NO:151, SEQ ID NO:163, SEQ ID NO:199, and SEQ ID NO:211; a CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:8, SEQ ID NO:20, SEQ ID NO:44, SEQ ID NO:56, SEQ ID NO:68, SEQ ID NO:80, SEQ ID NO:104, SEQ ID NO:116, SEQ ID NO:128, SEQ ID NO:140, SEQ ID NO:152, SEQ ID NO:164, SEQ ID NO:200 and SEQ ID NO:212; and a CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:9, SEQ ID NO:21, SEQ ID NO:45, SEQ ID NO:57, SEQ ID NO:69, SEQ ID NO:81, SEQ ID NO:105, SEQ ID NO:117, SEQ ID NO:129, SEQ ID NO:141, SEQ ID NO:153, SEQ ID NO:165, SEQ ID NO:201 and SEQ ID NO:213; The light chain variable region (ii) a CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:22, SEQ ID NO:46, SEQ ID NO:58, SEQ ID NO:70, SEQ ID NO:82, SEQ ID NO:106, SEQ ID NO:118, SEQ ID NO:130, SEQ ID NO:142, SEQ ID NO:154, SEQ ID NO:166, SEQ ID NO:202, and SEQ ID NO:214; a CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:11, SEQ ID NO:23, SEQ ID NO:47, SEQ ID NO:59, SEQ ID NO:71, SEQ ID NO:83, SEQ ID NO:107, SEQ ID NO:119, SEQ ID NO:131, SEQ ID NO:143, SEQ ID NO:155, SEQ ID NO:167, SEQ ID NO:203 and SEQ ID NO:215; and The CDR3 may comprise or consist of an amino acid sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:24, SEQ ID NO:48, SEQ ID NO:60, SEQ ID NO:72, SEQ ID NO:84, SEQ ID NO:108, SEQ ID NO:120, SEQ ID NO:132, SEQ ID NO:144, SEQ ID NO:156, SEQ ID NO:168, SEQ ID NO:204 and SEQ ID NO:216.

[0045] In another embodiment, the heavy chain variable region comprises: (i) a CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 43, SEQ ID NO: 79, SEQ ID NO: 115, and SEQ ID NO: 211; a CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:44, SEQ ID NO:80, SEQ ID NO:116, and SEQ ID NO:212; and a CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 45, SEQ ID NO: 81, SEQ ID NO: 117, and SEQ ID NO: 213; The light chain variable region (ii) a CDR1 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 82, SEQ ID NO: 118, and SEQ ID NO: 214; a CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:47, SEQ ID NO:83, SEQ ID NO:119, and SEQ ID NO:215; and It may comprise a CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:48, SEQ ID NO:84, SEQ ID NO:120, and SEQ ID NO:216.

[0046] In another embodiment, the heavy chain variable region comprises a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 43, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 44, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 45, and the light chain variable region comprises a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 46, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 47, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 48.

[0047] In another embodiment, the heavy chain variable region comprises a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 79, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 80, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 81, and the light chain variable region comprises a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 82, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 83, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 84.

[0048] In another embodiment, the heavy chain variable region comprises a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 115, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 116, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 117, and the light chain variable region comprises a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 118, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 119, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 120.

[0049] In another embodiment, the heavy chain variable region comprises a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 211, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 212, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 213, and the light chain variable region comprises a CDR1 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 214, a CDR2 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 215, and a CDR3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 216.

[0050] In another embodiment, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 303, 307, 311, 315, 319, 323, 327, 331, 335, 339, 343, 347, 351, 355, 359, 363, 367, 371, 375, 379, 383, 387, 391, 395, and 399, and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 308, 312, 316, 320, 324, 328, 332, 336, 340, 344, 348, 352, 356, 360, 364, 368, 372, 376, 380, 384, 388, 392, 396, and 400.

[0051] In another embodiment, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 315, 327, 339, and 371, and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 316, 328, 340, and 372. Preferably, the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 315 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 316; the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 327 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 328; the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 339 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 340; or the heavy chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 371 and the light chain variable region comprises the amino acid sequence set forth in SEQ ID NO: 372.

[0052] In another aspect, there is provided an anti-CD300c monoclonal antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region including CDR1 to CDR3 that contain or are composed of amino acid sequences represented by the following formulas (1) to (3), respectively, and a light chain variable region including CDR1 to CDR3 that contain or are composed of amino acid sequences represented by the following formulas (4) to (6), respectively (each amino acid sequence is in the N→C orientation):

[0053] FTFX1X2X3X4MX5WVR (1) (sequence number 403) In the above formula, X1= G or S X2= S, R or D X3= N or Y X4 = Y, A, G or H X5 = S or H X1ISX2SGX3X4TYYAX5 (2) (SEQ ID NO: 404) In the above formula, X1 = T or A X2 = G or S X3 = T or G X4 = S or Y X5= D or E YCAX1X2X3X4X5X6X7X8X9W (3) (SEQ ID NO: 405) In the above formula, X1= R or S X2 = G or S X3= M, S, Y or I X4= W, Q, G or R X5= G or L X6= M, I or P X7= D, F or L X8= V or D X9 = I, Y or absent CX1X2X3X4X5X6X7X8X9X10X11VX12W (4) (SEQ ID NO: 406) In the above formula, X1= T or S X2 = G or R X3= K, N or S X4= H, N or S X5= R, I or G X6 = H, G or I X7= T, I or S X8= R, A, K, or absent X9= R, S, G, or absent X10 = N or absent X11 = Y or absent X12 = N, H or Q X1X2X3X4RPSGVX5 (5) (SEQ ID NO: 407) In the above formula, X1= L, S, R or E X2= D, K or N X3 = S or N X4= E, N, Q or K X5= P or R YCX1X2X3X4X5X6X7X8X9X10VF (6) (sequence number 408) In the above formula, X1 = Q, A, or S X2 = S or A X3= Y or W X4 = D or A X5= S, D or G X6= S, N or T X7= S, L, N or K X8= V, S, N or G X9= G, L, V or absent X10 = P or absent.

[0054] In certain embodiments, the anti-CD300c antibody or antigen-binding fragment may comprise a sequence having 80% or more, preferably 90% or more, more preferably 95% or more, and most preferably 98% or more sequence identity with the CDR sequences or the sequences set out in Tables 3, 4 and 5 below.

[0055] In certain embodiments, amino acid sequence variants of the antibodies of the present invention are contemplated. For example, improving the binding affinity and / or other biological properties of the antibody may be desirable. Amino acid sequence variants of the antibody can be produced by introducing appropriate modifications into the nucleotide sequence encoding the molecule or by peptide synthesis. Such modifications include, for example, deletion of residues from the antibody's amino acid sequence, and / or insertion and / or substitution of residues within such amino acid sequences. Any combination of various changes, including deletion, insertion, and substitution, can be made to arrive at the final construct, provided that the final construct retains the desired properties, e.g., antigen-binding properties. Sites of interest for substitution mutagenesis include the heavy chain variable region (HVR) and framework regions (FR). Conservative substitutions are provided in Table 1 under the heading "Preferred Substitutions" and are further described below in connection with amino acid side chain classes (1) through (6). Amino acid substitutions can be introduced into the molecule of interest and the products screened for the desired activity, e.g., maintained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0056] [Table 1-1]

[0057] [Table 1-2]

[0058] Amino acids can be grouped according to their common side chain properties as follows: (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0059] Non-conservative substitutions involve exchanging one member of such a class for another class.

[0060] As used herein, the term "amino acid sequence variant" includes substantial variants in which amino acid substitutions are made at one or more hypervariable region residues of a parent antibody-binding molecule (e.g., a humanized or human antibody). Generally, the resulting variants selected for further study have / have altered, e.g., improved, specific biological properties (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody-binding molecule, or substantially retain the specific biological properties of the parent antigen-binding molecule. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage display-based affinity maturation techniques known in the art. Briefly, one or more HVR residues are mutated, and the mutant antigen-binding molecules are displayed on phage and screened for a specific biological activity (e.g., binding affinity). In certain embodiments, substitutions, insertions, or deletions are made within one or more HVRs, so long as such changes do not substantially reduce the ability of the antigen-binding molecule to bind to antigen. For example, conservative changes (eg, conservative substitutions as provided herein) can be made in HVRs that do not substantially reduce binding affinity.

[0061] Amino acid sequence insertions can include amino- and / or carboxyl-terminal fusions ranging in length from a single residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion includes an antibody with an N-terminal methionyl residue. Other insertional variants of the molecule can include the N- or C-terminal fusion of a polypeptide that increases the serum half-life of the antibody. Still other insertional variants of the molecule can include the N- or C-terminal fusion of a polypeptide that facilitates crossing the blood-brain barrier (BBB).

[0062] Also provided are variants of the antibody or antigen-binding fragment thereof of the present invention that have improved affinity for the CD300c antigen. Such variants can be generated by CDR mutations (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et al., Bio / Technology, 10, 779-783, 1992), the use of mutator strains of E. coli (Low et al., J. Mol. Biol., 250, 359-368, 1996), DNA shuffling (Patten et al., Curr. Opin. Biotechnol., 8, 724-733, 1997), phage display (Thompson et al., J. Mol. Biol., 256, 77-88, 1996), and sexual PCR (Crameri et al., J. Mol. Biol., 256, 77-88, 1996). These affinity maturation protocols can be obtained by a number of methods, including those described in [Verhoeyen et al., Science, 239, 1534-1536, 1988].

[0063] In one embodiment, the anti-CD300c monoclonal antibody or antigen-binding fragment thereof may have interspecies cross-reactivity. Specifically, the anti-CD300c monoclonal antibody or antigen-binding fragment thereof may be cross-reactive with both human and mouse CD300c antigens. Such cross-reactivity is confirmed in Experimental Examples 4.1 to 4.4.

[0064] In other embodiments, the anti-CD300c monoclonal antibody or antigen-binding fragment thereof may be in the form of an antibody-drug conjugate conjugated with another drug, or may be provided in such a form.

[0065] The term "antibody-drug conjugate" as used herein refers to a form in which an antibody and a drug are chemically linked together without reducing the biological activity of the antibody and the drug. The antibody-drug conjugate as used herein refers to a form in which a drug is conjugated to the N-terminal amino acid residue of the heavy and / or light chain of the antibody, specifically, to the α-amine group at the N-terminus of the heavy and / or light chain of the antibody.

[0066] The term "drug" refers to any substance having a specific biological activity on cells (e.g., cancer cells), and includes DNA, RNA, and peptides. The drug may be in a form containing a reactive group capable of reacting with an α-amine group to crosslink, or may be in a form to which a linker containing a reactive group capable of reacting with an α-amine group to crosslink is connected.

[0067] The reactive group capable of reacting with an α-amine group to crosslink is not particularly limited as long as it can react with the N-terminal α-amine group of an antibody heavy or light chain to crosslink, and includes all types known in the art that react with an amine group, including, but not limited to, isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, anhydride, and fluorophenyl ester.

[0068] The drug may be any drug, regardless of type, that can treat the disease targeted by the anti-CD300c antibody or antigen-binding fragment thereof according to the present invention, and may preferably be an anticancer drug.

[0069] Nucleic acids, vectors, host cells and production methods The anti-CD300c monoclonal antibody or antigen-binding fragment thereof of the present invention can be produced by any antibody production technique known in the art.

[0070] According to another aspect of the present invention, there is provided a nucleic acid molecule (e.g., polynucleotide) encoding the anti-CD300c monoclonal antibody or antigen-binding fragment thereof. Such a nucleic acid molecule may encode an amino acid sequence including the heavy chain variable region or heavy chain CDR region and / or the light chain variable region or light chain CDR region of the anti-CD300c monoclonal antibody. The sequences of nucleic acid molecules encoding the heavy and light chain variable regions and CDR regions of the anti-CD300c monoclonal antibody according to the present invention can be found in Tables 3 to 5 and Figure 1.

[0071] The term "nucleic acid molecule" encompasses DNA (gDNA and cDNA) and RNA molecules. Nucleotides, the basic building blocks of nucleic acid molecules, include not only naturally occurring nucleotides but also analogs with modified sugars or bases. The sequences of the nucleic acid molecules encoding the heavy chain variable region, light chain variable region, and CDR regions of the present invention may be modified. Such modifications include nucleotide addition, deletion, or non-conservative or conservative substitution. The nucleic acid molecules of the present invention are also understood to include nucleotide sequences that are substantially identical to the nucleotide sequences described above. "Substantial identity" refers to a nucleotide sequence that is 80% or more identical, in one specific example, 90% or more identical, in another specific example, 95% or more identical, and in another specific example, 98% or more identical when the nucleotide sequence of the present invention is aligned with any other sequence for maximum correspondence and the aligned sequences are analyzed using an algorithm commonly used in the art.

[0072] Another aspect of the present invention provides one or more vectors (eg, expression vectors) containing the nucleic acid.

[0073] The term "vector" refers to a nucleic acid molecule capable of transporting other nucleic acids linked thereto. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be inserted. Another type of vector is a viral vector, in which virally derived DNA or RNA sequences are present in the vector for packaging into the virus. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, thereby being replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Common expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, "plasmid" and "vector" may be used interchangeably, as the plasmid is the most commonly used form of vector. However, the invention includes other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0074] According to another aspect of the invention, host cells are provided that contain one or more nucleic acid molecules (eg, polynucleotides) encoding the monoclonal antibodies of the invention.

[0075] The host cell may be a cell transformed with the recombinant vector of the present invention. Any host cell known in the art that allows stable and continuous cloning and expression of the recombinant vector may be used. Suitable prokaryotic host cells include Escherichia coli, Bacillus species and strains such as Bacillus subtilis and B. thuringensis, Enterobacteriaceae and strains such as Salmonella typhimurium and Serratia marcescens, and various Pseudomonas species. Suitable eukaryotic host cells for transformation include yeast, e.g., Saccharomyces cerevisiae, insect cells, plant cells, and animal cells, e.g., Sp2 / 0, Chinese hamster ovary (CHO) K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, and MDCK cell lines. The term "host cell" also refers to a transformed cell or a cell capable of expressing a selected gene of interest after transformation with a nucleic acid sequence. The term also includes the progeny of a mother cell, regardless of whether the progeny are identical in morphology or genetic make-up to the original parent, so long as the selected gene is present.

[0076] Another aspect of the present invention provides a method for producing an anti-CD300c monoclonal antibody or an antigen-binding fragment thereof, comprising the step of culturing the host cell.

[0077] Host cells for producing the antibody or antigen-binding fragment thereof are cultured in appropriate media and under appropriate conditions known in the art. Such culture processes can be easily adjusted and used by those skilled in the art depending on the host cells selected. Culture processes are classified into suspension culture and adherent culture depending on the type of cell growth, and into batch, fed-batch, and continuous culture depending on the culture type. Various culture processes are disclosed, for example, in "Biochemical Engineering" by James M. Lee, Prentice-Hall International Editions, pp. 138-176.

[0078] To recover the antibody, any method known in the art for the purification of immunoglobulins can be used, for example, chromatography (ion exchange, affinity (e.g., protein A), size exclusion, etc.), centrifugation, differential solubility, or other standard techniques for purifying proteins.

[0079] Combination with immunotherapy The present inventors have confirmed that an anti-CD300c antibody or antigen-binding fragment thereof exhibits enhanced anti-cancer effects when used in combination with one or more other immunological anti-cancer agents. Therefore, the anti-CD300c antibody or antigen-binding fragment thereof of the present invention can be used in combination with one or more other immunological anti-cancer agents for the prevention or treatment of cancer.

[0080] Immunotherapy for cancer has a novel mechanism for activating the body's immune cells to kill cancer cells, and thus has the advantage of being widely used for many cancers, even in the absence of specific genetic mutations. Furthermore, immunotherapy for cancer treats cancer by strengthening the patient's own immune system, resulting in fewer side effects and significantly improving the patient's quality of life and prolonging survival. Such immunotherapy for cancer includes immune barrier inhibitors and may be manufactured by known methods or commercially available products. Examples of immunotherapy for cancer include, but are not limited to, anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-CD47, anti-KIR, anti-LAG3, anti-CD137, anti-OX40, anti-CD276, anti-CD27, anti-GITR, anti-TIM3, anti-41BB, anti-CD226, anti-CD40, anti-CD70, anti-ICOS, anti-CD40L, anti-BTLA, anti-TCR, and anti-TIGIT antibodies. Additionally, examples of immunological anti-cancer agents include, but are not limited to, durvalumab (Imfinzi), atezolizumab (Tecentriq), avelumab (Bavencio), pembrolizumab (Keytruda), nivolumab (Opdivo), αCD47, cemiplimab (Libtayo), magrolimab (Hu5F9-G4), and ipilimumab (Yervoy).

[0081] In one embodiment, the immunological anti-cancer agent may comprise one or more selected from the group consisting of anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-CD47, anti-KIR, anti-LAG3, anti-CD137, anti-OX40, anti-CD276, anti-CD27, anti-GITR, anti-TIM3, anti-41BB, anti-CD226, anti-CD40, anti-CD70, anti-ICOS, anti-CD40L, anti-BTLA, anti-TCR, and anti-TIGIT antibodies. For example, the immunological anti-cancer agent may comprise one or more selected from the group consisting of anti-PD-1, anti-PD-L1, anti-CTLA-4, and anti-CD47 antibodies.

[0082] In other embodiments, the immunosuppressant may include one or more selected from the group consisting of durvalumab (Imfinzi), atezolizumab (Tecentriq), pembrolizumab (Keytruda), nivolumab (Opdivo), αCD47, and ipilimumab (Yervoy).

[0083] Methods for preventing or treating cancer Another aspect of the present invention provides a method for preventing or treating cancer in a subject, ameliorating or reducing the severity of at least one symptom or sign of cancer, inhibiting metastasis, or inhibiting cancer growth using an anti-CD300c antibody or antigen-binding fragment thereof according to the present invention. As used herein, "preventing or treating cancer" includes inhibiting cancer growth, survival, metastasis, recurrence, or resistance to anticancer drugs. Such a method may comprise administering an anti-CD300c antibody or antigen-binding fragment thereof according to the present invention to a subject in need of cancer prevention or treatment.

[0084] As used herein, the term "cancer" refers to a physiological condition in mammals that is typically characterized by unregulated cell growth. Cancers that are the subject of prevention or treatment in the present invention may include, depending on their site of origin, colorectal cancer, small intestine cancer, rectum cancer, colon cancer, thyroid cancer, endocrine gland cancer, oral cancer, tongue cancer, pharynx cancer, larynx cancer, esophageal cancer, cervical cancer, uterine cancer, fallopian tube cancer, ovarian cancer, brain cancer, head and neck cancer, lung cancer, lymph node cancer, gallbladder cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer (or melanoma), breast cancer, stomach cancer, bone cancer, and blood cancer, but may also include all cancers as long as they express CD300c protein on the surface of cancer cells. In one embodiment, the cancer may include any one or more selected from the group consisting of colorectal cancer, colon cancer, thyroid cancer, oral cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, brain cancer, lung cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, tongue cancer, breast cancer, uterine cancer, stomach cancer, bone cancer, and blood cancer. In another embodiment, the cancer may be a solid cancer.

[0085] In one embodiment, the method may further comprise determining the expression level of CD300c protein based on a biological sample or specimen from the subject prior to administration of the anti-CD300c antibody or antigen-binding fragment thereof.

[0086] The method may also include a step of determining that a subject is suitable for treatment using an anti-CD300c antibody or its antigen-binding fragment when the CD300c protein expression level determined using the subject's biological sample or specimen is equal to or higher than a certain level. Specifically, the method may include a step of determining that a subject is suitable for treatment using an anti-CD300c antibody or its antigen-binding fragment when the CD300c protein expression level determined using the subject's biological sample or specimen is statistically significantly higher (e.g., 10% or higher) than a control group (e.g., the expression level in normal individuals without cancer or the average expression level in cancer patients). However, the differences in CD300c protein expression level shown above are merely exemplary and may be, but are not limited to, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, or 70% or more. Preferably, the control group may be the average expression level in patients with the same type of cancer.

[0087] In one embodiment of the present invention, overall survival was compared according to CD300c expression level using data from kidney cancer (530 patients), pancreatic cancer (177 patients), and liver cancer (370 patients) patients obtained from the US TCGA (The Cancer Genome Atlas) database. The results showed that cancer patients with higher CD300c expression levels than the average CD300c expression level for each cancer type showed shorter overall survival than patients without such levels. Therefore, in order to increase the therapeutic response rate of a subject to an anti-CD300c antibody, it may be preferable to refer to the expression level of the CD300c protein in the subject.

[0088] In another embodiment, the method may further comprise administering one or more immunological anti-cancer agents. When (i) an anti-CD300c antibody or antigen-binding fragment thereof is used in combination with (ii) one or more immunological anti-cancer agents, (i) and (ii) may be administered simultaneously or sequentially.

[0089] "Sequential administration" means that one component is administered, followed by another component immediately after or at a certain interval thereafter. The components can be administered in any order. That is, one or more immunological anticancer agents can be administered immediately after or at a certain interval after the administration of an anti-CD300c antibody or antigen-binding fragment thereof, or vice versa. Alternatively, one of the one or more immunological anticancer agents can be administered first, followed by the anti-CD300c antibody or antigen-binding fragment thereof, followed by another of the one or more immunological anticancer agents.

[0090] In another embodiment, the anti-CD300c antibody or antigen-binding fragment thereof may be administered together with two or more immunological anti-cancer agents. For example, it has been shown that the greatest cancer cell proliferation inhibitory effect can be achieved when the anti-CD300c antibody or antigen-binding fragment thereof is administered together with two immunological anti-cancer agents (e.g., an anti-PD-L1 antibody and an anti-PD-1 antibody, or an anti-PD-1 antibody and an anti-CTLA-4 antibody).

[0091] The antibody or antigen-binding fragment thereof according to the present invention, and optionally one or more additional anti-cancer agents, can each be administered in various ways depending on whether local or systemic treatment is desired and the area to be treated. The method of administering these components to a subject can vary depending on the purpose of administration, the site of disease, the condition of the subject, etc. The route of administration may be oral, parenteral, inhalation, topical, or local (e.g., intralesional) administration. For example, parenteral administration can include, but is not limited to, intravenous, subcutaneous, intraperitoneal, intrapulmonary, intraarterial, intramuscular, rectal, intravaginal, intraarticular, intraprostatic, intranasal, intraocular, intravesical, intraspinal, or intraventricular (e.g., intracerebroventricular) administration. Furthermore, when used in combination, the anti-CD300c antibody and the additional immunosuppressant can be administered via the same route or via different routes.

[0092] In the above-described method, the effective dose of the anti-CD300c antibody or antigen-binding fragment thereof according to the present invention and, optionally, one or more additional anticancer agents varies depending on the age, sex, and weight of the individual (patient), and is generally about 0.01 mg to 100 mg, or 5 mg to about 50 mg per kg of body weight, administered once or several times a day. However, the scope of the present invention is not limited to these amounts, as they may vary depending on the route and duration of administration, severity of the disease, sex, weight, age, etc.

[0093] The method according to the present invention may include a step of determining the expression level of CD300c protein in advance in a subject, and whether or not to administer an anti-CD300c antibody or an antigen-binding fragment thereof can be determined based on the expression level.

[0094] In another embodiment, the method of the present invention may include a step of selecting additional (one or more) immunological anti-cancer agents suitable for use in combination with the anti-CD300c antibody or its antigen-binding fragment by administering the anti-CD300c antibody or its antigen-binding fragment to a subject and then measuring changes in the expression levels of specific markers.

[0095] Specifically, the method may further include determining the expression level of one or more markers selected from the following markers using a biological sample or specimen from the subject to which the anti-CD300c antibody or antigen-binding fragment thereof has been administered:

[0096] Bst2, Cd40, Cd70, Cd86, Ccl8, Xcl1, Ccr7, Cd80, Cd206, Msr1, Arg1, Vegfa, Pdgfrb, C ol4a1, Hif1a, Vcam1, Icam1, Gzma, Gzmb, Icos, Cd69, Ifng, Tnf, Cd1d1, Cd1d2, Cd38, Cxcr6, Xcr1, Tbx21, Stat1, Stat4, Cxcr3, IL-12b, IL-4, IL-6, IL-13, PD-1, PD-L1, CTLA-4, Lag3, Tim3, Ox40, Gitr, Hvem, CD27, CD28, Cma1, Timd4, Bcl6, Cxcl5 and Ccl21a.

[0097] See Table 2 below for a description of the markers.

[0098] [Table 2-1]

[0099] [Table 2-2]

[0100] [Table 2-3]

[0101] [Table 2-4]

[0102] [Table 2-5]

[0103] [Table 2-6]

[0104] [Table 2-7]

[0105] [Table 2-8]

[0106] In another embodiment, the method may further include selecting an additional immunosuppressant based on the expression levels of the identified markers. In this case, the markers may include, but are not limited to, PD-1, PD-L1, CTLA-4, Lag3, Tim3, Icos, Ox40, Gitr, Hvem, CD27, and CD28. In another embodiment, the markers may include one or more selected from the group consisting of PD-1, PD-L1, CTLA-4, Lag3, Tim3, Icos, Ox40, Gitr, Hvem, CD27, and CD28. Preferably, the markers may include one or more selected from the group consisting of PD-1, PD-L1, CTLA-4, Lag3, and Tim3. Additionally, the markers may include one or more selected from the group consisting of Icos, Ox40, Gitr, Hvem, CD27, and CD28.

[0107] Changes in the expression levels of such markers refer to changes in tumor / immune-related markers that affect the tumor-suppressing effect observed when the anti-CD300c antibody or its antigen-binding fragment of the present invention is administered to an individual. For example, changes in the expression levels of such markers can include changes in the expression patterns of protein markers associated with the activity of immune cells (e.g., dendritic cells, macrophages, T cells, and NKT cells), immune barrier protein markers, tumor microenvironment (TME) protein markers that affect tumor growth, and markers associated with Th1 and Th2 responses. For specific examples of markers, see the above description. Changes in such expression patterns can be used to predict the probability of a patient's response to a drug or to select anticancer drugs, including other immune barrier inhibitors, that maximize anticancer effects. Furthermore, these markers can be used to determine whether a patient is suitable for treatment with the antibody, monitor the efficacy of drug treatment, and provide information for treatment methods, including drug dosage, administration, and combination therapy.

[0108] According to an example of the present invention, it was confirmed that an enhanced anti-tumor effect was achieved when the anti-CD300c antibody or antigen-binding fragment thereof of the present invention was administered in combination with other immune barrier inhibitors (one or more of the anti-PD-L1 antibody durvalumab (Imfinzi), the anti-PD-1 antibody nivolumab (Opdivo), the anti-PD-1 antibody pembrolizumab (Keytruda), the anti-CTLA-4 antibody, and the anti-CD47 antibody (αCD47)) selected based on changes in the expression levels of the markers observed in individuals.

[0109] In another embodiment, the method may further include confirming the therapeutic responsiveness of the anti-CD300c antibody or antigen-binding fragment thereof based on the expression levels of the identified markers, which may include, but are not limited to, vegfa, pdgfrb, Col4a1, Hif1a, Bst2, CCL8, Xcl1, CCR7, CD80, Tbx21, Stat1, Stat4, Ifng, Cxcr3, IL-6, Gzma, Icos, Cd69, Cd1d1, Cd38, Cxcr6, Ox40, Gitr, CD27, and CD28. Preferably, the markers may include one or more selected from the group consisting of vegfa, pdgfrb, Col4a1, Hif1a, Bst2, CCL8, Xcl1, CCR7, CD80, Tbx21, Stat1, Stat4, Ifng, Cxcr3, IL-6, Gzma, Icos, Cd69, Cd1d1, Cd38, and Cxcr6. Alternatively, the markers may include one or more selected from the group consisting of vegfa, pdgfrb, Col4a1, and Hif1a. Alternatively, the markers may include one or more selected from the group consisting of Bst2, CCL8, and Xcl1. In another embodiment, the markers may include CCR7, CD80, or a combination thereof. Alternatively, the markers may include one or more selected from the group consisting of Tbx21, Stat1, Stat4, Ifng, Cxcr3, and IL-6.

[0110] In another embodiment, the method may further include determining that the therapeutic responsiveness of the anti-CD300c antibody or its antigen-binding fragment is good or excellent if the expression level of one or more of the markers is reduced compared to a subject not administered with the anti-CD300c antibody or its antigen-binding fragment. For example, the method may determine that the therapeutic responsiveness of the anti-CD300c antibody or its antigen-binding fragment is good or excellent if the expression level of one or more markers selected from the group consisting of vegfa, pdgfrb, Col4a1, Hif1a, and IL-6 is reduced compared to a subject not administered with the anti-CD300c antibody or its antigen-binding fragment. In this case, a decrease in expression level refers to a statistically significant decrease, and the percentage decrease in expression level may include, but is not limited to, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, or about 100% or more.

[0111] Furthermore, the method may further include determining that the therapeutic responsiveness of the anti-CD300c antibody or its antigen-binding fragment is good or excellent if the expression level of one or more of the markers is increased compared to a subject not administered with the anti-CD300c antibody or its antigen-binding fragment. For example, the method may determine that the therapeutic responsiveness of the anti-CD300c antibody or its antigen-binding fragment is good or excellent if the expression level of one or more markers selected from the group consisting of Bst2, CCL8, Xcl1, CCR7, CD80, Tbx21, Stat1, Stat4, Ifng, Cxcr3, Gzma, Icos, Cd69, Cd1d1, Cd38, Cxcr6, Ox40, Gitr, Cd27, and Cd28 is increased compared to a subject not administered with the anti-CD300c antibody or its antigen-binding fragment. In this case, an increase in expression level means a statistically significant increase, and the increase rate of expression level may include, but is not limited to, about 10% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, and about 100% or more.

[0112] Pharmaceutical Composition Another aspect of the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising an anti-CD300c antibody or an antigen-binding fragment thereof as an active ingredient.

[0113] The anti-CD300c antibody or antigen-binding fragment thereof may be contained in a composition in a prophylactically or therapeutically effective amount, and the pharmaceutical composition may be administered to a subject to inhibit cancer growth, survival, metastasis, recurrence, or resistance to anticancer drugs.

[0114] In one embodiment, the pharmaceutical composition may further comprise one or more immunological anti-cancer agents. Specifically, the anti-CD300c antibody or antigen-binding fragment thereof and, optionally, the additional immunological anti-cancer agent may be contained in the same composition or in separate compositions. When contained in separate compositions, the anti-CD300c antibody or antigen-binding fragment thereof and the additional immunological anti-cancer agent may be formulated separately and administered simultaneously or sequentially.

[0115] To prepare the pharmaceutical composition of the present invention, the antibody or antigen-binding fragment thereof and, optionally, an additional immunosuppressant can be mixed with a pharmaceutically acceptable carrier and / or excipient. The pharmaceutical composition can be prepared in the form of a lyophilized formulation or an aqueous solution. See, for example, Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984).

[0116] Acceptable carriers and / or excipients (including stabilizers) are non-toxic to a subject at the dosages and concentrations employed and include buffers (e.g., phosphate, citrate, or other organic acids); antioxidants (e.g., ascorbic acid or methionine); preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens, e.g., methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (about 10 or fewer residues) polypeptides; proteins (e.g., serum albumin, gelatin, or immunoglobulin; hydrophilic polymers (e.g., polyvinylpyrrolidone); amino acids (e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates, such as glucose, mannose, or dextrin; chelating agents (e.g., EDTA); sugars (e.g., sucrose, mannitol, trehalose, or sorbitol); counterions for salt formation (e.g., sodium); metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants (e.g., TWEEN®, PLURONICS®, or polyethylene glycol (PEG)).

[0117] The pharmaceutical composition of the present invention can be formulated in a suitable form known in the art depending on the route of administration.

[0118] As used herein, the term "prophylactically or therapeutically effective amount" or "effective amount" refers to the amount of an active ingredient in a composition that is effective in preventing or treating cancer in a subject, and refers to an amount that is sufficient to prevent or treat cancer at a reasonable benefit / risk ratio applicable to any medical treatment, but does not cause side effects. The level of the effective amount can be determined based on factors including the patient's health condition, type and severity of the disease, drug activity, sensitivity to the drug, administration method, administration time, administration route and excretion rate, treatment duration, coadministered or concomitant drugs, and other factors well known in the medical field. In this regard, it is important to administer an amount that will achieve maximum effect with minimal side effects or minimal amount without side effects, taking all of the above factors into consideration, and this can be easily determined by one of ordinary skill in the art.

[0119] Specifically, the effective amount of each active ingredient in the pharmaceutical composition of the present invention varies depending on the age, sex, and weight of the individual (patient), and generally ranges from about 0.01 mg to 100 mg, or 5 mg to about 50 mg per kg of body weight, and can be administered once or several times a day. However, since the amount may increase or decrease depending on the route and duration of administration, severity of the disease, sex, weight, age, etc., the scope of the present invention is not limited to these amounts.

[0120] Cancer prevention or treatment kits Another aspect of the present invention provides a kit for preventing or treating cancer, comprising a composition containing an anti-CD300c antibody or antigen-binding fragment thereof according to the present invention and instructions for use of the antibody or antigen-binding fragment thereof, wherein the composition may contain a prophylactically or therapeutically effective amount of the anti-CD300c antibody or antigen-binding fragment thereof.

[0121] In one embodiment, the instructions may include instructions directing the use of the antibody or antigen-binding fragment thereof in combination with one or more additional anti-cancer agents.

[0122] In one embodiment, the instructions may include instructions for dosing or administering the active ingredient. For example, the instructions may include instructions for measuring the expression level of CD300c protein using a biological sample or specimen obtained from the subject prior to administration of the antibody or antigen-binding fragment thereof. Optionally, the kit may include a device or apparatus necessary for administering the active ingredient(s).

[0123] Dosage Effective or non-toxic amounts of the anti-CD300c antibody or antigen-binding fragment thereof according to the present invention, and optionally, the additional immunological anti-cancer agent, can be determined by routine experimentation. For example, the therapeutically active amount of the antibody or immunological anti-cancer agent will vary depending on factors such as the stage of the disease, the severity of the disease, the subject's age, sex, medical complications, and weight, the ability of the components to elicit the desired response in the subject, and the dosage of the concomitant anti-cancer agent. The dosage and administration regimen of the anti-CD300c antibody or antigen-binding fragment thereof or the additional immunological anti-cancer agent can be adjusted to provide the optimal therapeutic response. For example, several divided doses can be administered daily, weekly, every two weeks, every three weeks, every four weeks, etc., and the dosage can be proportionally reduced or increased depending on the exigencies of the therapeutic situation.

[0124] Method and kit for providing information for cancer prevention or treatment In another aspect of the present invention, there is provided a method for providing information for the prevention or treatment of cancer, comprising determining the expression level of CD300c protein using a biological sample or specimen obtained from a subject in need of cancer prevention or treatment. The method can also be used for pre-screening for administration of an anti-CD300c antibody or its antigen-binding fragment.

[0125] In one embodiment, the step of determining the expression level of the CD300c protein (marker) may include using a molecule that specifically binds to the CD300c protein, such as an antibody, a substrate, a ligand, or a cofactor, or a molecule that specifically binds to the CD300c mRNA, such as a primer pair or a probe. Methods for determining expression levels using these reagents, including those described above, are well known in the art.

[0126] In one embodiment, the method may include determining that a subject is suitable for treatment using an anti-CD300c antibody or its antigen-binding fragment when the expression level of CD300c protein determined using the subject's biological sample or specimen is equal to or greater than a certain level. Specifically, the method may include determining that a subject is suitable for treatment using an anti-CD300c antibody or its antigen-binding fragment when the expression level of CD300c protein determined using the subject's biological sample or specimen is statistically significantly higher (e.g., 10% or higher) than a control group (e.g., the expression level in normal individuals without cancer or the average expression level in cancer patients). However, the above-mentioned differences in CD300c protein expression level are merely exemplary and may be, but are not limited to, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, or 70% or more. Preferably, the control group may be the average expression level in patients with the same type of cancer.

[0127] In one embodiment of the present invention, overall survival was compared according to CD300c expression level using data from kidney cancer (530 patients), pancreatic cancer (177 patients), and liver cancer (370 patients) patients obtained from the US TCGA (The Cancer Genome Atlas) database. The results showed that cancer patients with higher CD300c expression levels than the average CD300c expression level for each cancer type showed shorter overall survival than patients without such levels. Therefore, in order to increase the therapeutic response rate of a subject to an anti-CD300c antibody, it may be preferable to refer to the expression level of the CD300c protein in the subject.

[0128] In other embodiments, the information for preventing or treating cancer may include, but is not limited to, information on one or more of therapeutic response to a therapeutic agent related to CD300c protein (e.g., an anti-CD300c antibody or its antigen-binding fragment), selection of a therapeutic agent, selection of a subject to be treated, prognosis of a subject, and survival time of a subject. Preferably, the information for preventing or treating cancer may include therapeutic response to an anti-CD300c antibody or its antigen-binding fragment, survival time of a subject, or both.

[0129] Another aspect of the present invention provides a kit for providing information for the prevention or treatment of cancer, comprising a substance for measuring the expression level of CD300c protein using a biological sample or specimen obtained from a subject in need of cancer prevention or treatment. The kit may also include one or more other component compositions, solutions, or devices suitable for the analytical method. The kit may be a kit for measuring the expression level of a protein marker, such as an enzyme-linked immunosorbent assay (ELISA) kit. The kit may also include other reagents required for immunological detection of the antibody and known in the art. The kit may further include a pharmaceutical composition comprising an anti-CD300c antibody or its antigen-binding fragment as an active ingredient.

[0130] The present invention will be described in more detail below with reference to the following examples, but the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0131] Example I. Production of anti-CD300c monoclonal antibody Example 1. Production of anti-CD300c monoclonal antibody Example 1.1. Construction of an anti-CD300c monoclonal antibody library To screen for anti-CD300c monoclonal antibodies, biopanning was performed using lambda, kappa, VH3VL1, and OPALTL phage libraries. Specifically, CD300c antigen was added to an immunotube at a concentration of 5 μg / mL and incubated for 1 hour to allow the antigen to adsorb to the surface of the tube. After that, 3% skim milk was added to suppress nonspecific reactions, and 10% of the CD300c antigen dispersed in the 3% skim milk was again adsorbed onto the surface of the tube. 12PFU of the antibody phage library was added to each immunoassay tube and allowed to bind to the antigen. After washing three times with TBST (tris-buffered saline-Tween 20) to remove nonspecifically bound phages, single-chain variable fragment (scFv) phage antibodies specifically bound to the CD300c antigen were eluted with 100 mM triethylamine. The eluted phages were neutralized with 1.0 M Tris-HCl buffer (pH 7.8) and then used to infect E. coli ER2537 at 37°C for 1 hour. The infected E. coli were plated on LB agar medium containing carbenicillin and cultured at 37°C for 16 hours. The formed E. coli colonies were then suspended in 3 mL of SB (super broth)-carbenicillin culture medium, and a portion was added with 15% glycerol and stored at -80°C until use, while the remainder was re-inoculated into SB-carbenicillin-2% glucose solution and cultured at 37°C. The resulting culture medium was centrifuged, and the supernatant containing phage particles was used for biopanning three times again to isolate and concentrate antigen-specific antibodies.

[0132] After three rounds of biopanning, E. coli containing the antibody genes were plated onto LB agar medium containing carbenicillin and cultured at 37°C for 16 hours. The resulting E. coli colonies were re-inoculated into SB-carbenicillin-2% glucose solution and cultured at 37°C until the absorbance (OD600nm) reached 0.5. IPTG was then added and the culture was continued for an additional 16 hours at 30°C. Periplasmic extraction was then performed, and a library pool of antibodies that specifically bind to the CD300c antigen was initially obtained from the results.

[0133] Example 1.2. Selection of anti-CD300c monoclonal antibodies To select anti-CD300c monoclonal antibodies with high binding affinity and specificity to the CD300c antigen, ELISA was performed using the library pool obtained by the same method as in Example 1.1. More specifically, CD300c antigen and CD300a antigen were dispensed into an ELISA plate at a concentration of 5 μg / mL per well in coating buffer (0.1 M sodium carbonate, pH 9.0) and incubated at room temperature for 3 hours to allow the antigens to bind to the plate. After washing three times with phosphate buffered saline-Tween 20 (PBST) to completely remove unbound antigen, 350 μL of PBST supplemented with 2% BSA (bovine serum albumin) was added to each well and incubated at room temperature for 1 hour. The plate was then washed again with PBST. Next, 25 μg of plasma membrane extract containing the scFv obtained by the same method as in Example 1.1 was added to each well and incubated at room temperature for 1 hour to allow binding to the antigen. After 1 hour, the plate was washed three times with PBST to remove unbound scFv, followed by the addition of 4 μg / mL of detection antibody and another 1 hour of incubation at room temperature. After the unbound detection antibody was removed with PBST, HRP-conjugated anti-rabbit IgG was added and incubated at room temperature for 1 hour, after which PBST was again used to remove unbound antibody. Next, 3,3',5,5'-tetramethylbenzidine (TMB) solution was added and incubated for 10 minutes to develop color. The color reaction was terminated by the addition of 2N sulfuric acid solution, and the absorbance was measured at 450 nm to confirm the antibody's specific binding to the CD300c antigen.

[0134] 1.3. Confirmation of anti-CD300c monoclonal antibody sequence The nucleotide sequences of the selected anti-CD300c monoclonal antibodies were confirmed using the same method as in Example 1.2. More specifically, plasmid DNA was extracted from the selected antibody clones using a plasmid miniprep kit, and DNA sequencing was performed to analyze the sequences of the complementarity-determining regions (CDRs). As a result, 25 anti-CD300c monoclonal antibodies, each with a different amino acid sequence, were isolated. The heavy and light chain variable regions of these 25 anti-CD300c monoclonal antibodies are shown in Tables 3 and 4 below.

[0135] [Table 3-1]

[0136] [Table 3-2]

[0137] [Table 3-3]

[0138] [Table 4-1]

[0139] [Table 4-2]

[0140] In each of the drawings referred to in Tables 3 and 4, the CDR regions (CDR1, CDR2, and CDR3) are underlined and indicated in order (i.e., CDR1 is indicated first, then CDR2, and then CDR3). The CDR regions included in each drawing are indicated by SEQ ID NOs as shown in Table 5 below:

[0141] [Table 5-1]

[0142] [Table 5-2]

[0143] [Table 5-3]

[0144] [Table 5-4]

[0145] [Table 5-5]

[0146] [Table 5-6]

[0147] [Table 5-7]

[0148] [Table 5-8]

[0149] As described above, 25 anti-CD300c monoclonal antibodies have been identified that have high binding affinity to the CD300c antigen and can be used for the prevention or treatment of cancers that specifically bind to the CD300c antigen.

[0150] Example 1.4. Production and purification of anti-CD300c monoclonal antibody Using the nucleotide sequence of the anti-CD300c monoclonal antibody identified in Example 1.3, we constructed expression vectors containing separate heavy and light chains capable of expressing the antibody. More specifically, we constructed the vectors by inserting genes into the pCIW3.3 vector to express the heavy and light chains, respectively, using the analyzed CDR sequences. The constructed heavy and light chain expression vectors were mixed with PEI (polyethylenimine) at a 1:1 mass ratio and transfected into 293T cells to induce antibody expression. On day 8, the culture medium was centrifuged to remove the cells, yielding a culture medium. The resulting culture medium was filtered and then resuspended in a solution containing 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 elution buffer (Thermofisher).

[0151] To confirm the antibody produced, 5 μg of purified antibody was added to reducing and non-reducing sample buffers, and electrophoresis was performed using a pre-made SDS PAGE (Invitrogen). The protein was then stained with Coomassie blue. The results under non-reducing conditions are shown in Figure 4, and those under reducing conditions in Figure 5.

[0152] As shown in Figures 4 and 5, it was confirmed that a highly pure anti-CD300c monoclonal antibody was produced and purified.

[0153] II. Expression of CD300c on cancer cells and binding of anti-CD300c monoclonal antibodies to the CD300c antigen Experimental Example 1. Expression of CD300c in cancer cells and immune cells Experimental Example 1.1. Confirmation of CD300c expression in cancer cell lines To evaluate the expression of CD300c in various cancer cells, we cultured various cell lines, including MKN45 (human gastric cancer cell line), IM95 (human gastric cancer cell line), HT-29 (human colon cancer cell line), A549 (human lung cancer cell line), HCT116 (human colon cancer cell line), MDA-MB-231 (human breast cancer cell line), and HepG2 (human liver cancer cell line), and assessed CD300c expression at the mRNA and protein levels. We also evaluated the expression of CD300c in the immune cell line THP-1 (human monocytic cell line). HEK293T (a general cell line) was used as a control.

[0154] Protein expression was confirmed using Western blot and flow cytometry (FACS) of fluorescently labeled cells. Specifically, each cultured cell line was fixed with 4% formaldehyde and blocked with 5% normal bovine serum albumin. Cells were then stained with 0.5 μg of eFluor 660-labeled anti-CD300c antibody (Invitrogen). Fluorescently labeled cells were then confirmed using flow cytometry (FACS).

[0155] As a result, we confirmed that CD300c antigen is expressed at the mRNA and protein levels in various cancer cells, including colon cancer, lung cancer, and breast cancer. Furthermore, as shown in Figure 6, analysis using flow cytometry (FACS) confirmed that CD300c is expressed at significantly higher levels in the human lung cancer cell line (A549) and the human monocyte cell line (THP-1) than in the general cell line (HEK293T).

[0156] Experimental Example 1.2. Confirmation of CD300c expression in cancer tissues and immune cells (I) To confirm the expression of CD300c in the patient's cancer tissue, a tissue microarray was performed as follows. The patient's colon cancer tissue was fixed in formalin and paraffin-blocked. Then, the tissue was sliced ​​into 2.0 mm diameter slices with a thickness of 3-5 μm for the microtissue array. The slices were then attached to slides in a uniform direction and allowed to dry. The cancer tissue was stained with H&E, followed by treatment with anti-CD300c antibody (Invitrogen) at a dilution of 1:500 to stain CD300c. As a result, as shown in Figure 7a, CD300c expression was confirmed in the patient's colon cancer tissue.

[0157] To confirm whether CD300c is expressed not only in colon cancer tissue but also in immune cells within cancer tissue, 2X10 5 CT26 cells were subcutaneously injected into 8-week-old BALB / c mice. Tumor tissue was collected from six control mice that had not received anti-CD300c antibody, sacrificed on day 25 (D25). Tumor tissue was then excised and incubated at 37°C for 1 hour in a mixture of collagenase D (20 mg / ml) and DNase I (2 mg / ml). The tissue was then filtered through a 70 μm cell strainer to lyse red blood cells, and then refiltered through a nylon mesh. The single-cell suspension was then blocked with CD16 / 32 antibody (Invitrogen) and stained with a cell viability stain (Invitrogen) and antibodies against total macrophage markers F4 / 80 (Abcam), CD11b (Abcam), CD11c (Abcam), CD3 (Abcam), CD4 (Thermofisher), CD8 (Thermofisher), and CD300c (Sino Biological). Data were then read using a CytoFLEX flow cytometer and analyzed using FlowJo software.

[0158] As a result, as shown in Figure 7b, we confirmed the presence of immune cells expressing CD300c in the tumor tissue of mice. These immune cells co-express both CD11b and CD11c markers, and examples of such cells include dendritic cells and macrophages. These results confirmed that CD300c is expressed in both cancer tissue and immune cells.

[0159] Experimental Example 1.3. Confirmation of CD300c expression in cancer tissues and immune cells (II) To confirm whether CD300c is expressed in human immune tissues and cancer cells, tissue microarrays were performed as follows. Normal tonsillar tissue and tissue from a colon cancer patient were fixed in formalin and then cut into paraffin blocks. The normal tonsillar tissue and tissue from a colon cancer patient were then selected for tissue microarray analysis. Then, the tissue was sliced ​​into 2.0 mm diameter slices with a thickness of 3-5 μm for microtissue array analysis. The slices were then attached to slides in a specific direction and allowed to dry. After staining the cancer tissue with H&E, CD300c was stained using anti-CD300c antibody (Invitrogen) at a dilution of 1:500.

[0160] As a result, as shown in Figures 8a and 8b, CD300c was confirmed to be expressed in both normal tonsillar tissue (Figure 8a), which is an immune tissue, and the patient's colon cancer tissue (Figure 8b). Because numerous immune cells, such as T cells and monocytes, are distributed in the tonsil, the expression of CD300c in tonsillar tissue indicates that CD300c is expressed in immune cells. Similar to Experimental Example 1.2, this experiment confirmed the expression of CD300c in the tissues of colon cancer patients. However, by observing CD300c expression in the tissues of all four colon cancer patients, it is significant that this experiment confirmed the expression of CD300c in a large number of colon cancer tissues.

[0161] Experimental Example 2. Confirmation of CD300c antigen recognition and binding by anti-CD300c monoclonal antibody Experimental Example 2.1. Confirmation of antigen binding affinity of anti-CD300c monoclonal antibody A binding ELISA was performed to confirm the antigen-binding ability of the anti-CD300c monoclonal antibody prepared in Example 1. Specifically, CD300c antigen (11832-H08H, Sino Biological) or CD300a antigen (12449-H08H, Sino Biological) was dispensed into an ELISA plate at a concentration of 8 μg / mL per well in coating buffer solution (0.1 M sodium carbonate, pH 9.0) and incubated at room temperature for 3 hours to allow the antigen to bind to the plate. After washing three times with PBST to completely remove unbound antigen, 300 μL of PBST supplemented with 5% BSA (bovine serum albumin) was added to each well and incubated at room temperature for 1 hour. The plate was then washed again with PBST. A 4-fold dilution of the anti-CD300c monoclonal antibody was then added and incubated at room temperature for 1 hour to allow binding to the antigen. After 1 hour, the cells were washed three times with PBST to remove unbound anti-CD300c monoclonal antibody, and then 4 μg / mL of a detection antibody (HRP-conjugated anti-Fc IgG) was added and incubated at room temperature for another 1 hour. After removing unbound detection antibody with PBST, TMB solution was added and incubated for 10 minutes to allow color development. The color development reaction was terminated by the addition of 2N sulfuric acid solution, and the absorbance was measured at 450 nm to confirm the antibody's specific binding to the CD300c antigen. The results are shown in Table 6 and Figure 9.

[0162] [Table 6]

[0163] As shown in Table 6, the EC50 (effective concentration of drug that causes 50% of the maximum response) of the anti-CD300c monoclonal antibodies was measured, and the binding affinity of all 14 clones, except for four clones (CK3, CL8, SK15, and SK16), was 0.2 μg / mL or less, confirming their high binding affinity. Furthermore, as shown in Figure 9, the sigmoid curve obtained from the binding ELISA results also confirmed that the anti-CD300c monoclonal antibodies of the present invention bind to the CD300c antigen with strong binding affinity.

[0164] Experimental Example 2.2. Confirmation of cellular antigen recognition by anti-CD300c monoclonal antibody To confirm that the anti-CD300c monoclonal antibody (CL7) recognizes the cellular antigen, FACS binding was performed.

[0165] CD300c was overexpressed in 293T cells (ATCC) and THP-1 cells (ATCC), and the cells were then cultured at 2 x 10 per microcentrifuge tube. 5 The cells were incubated with anti-CD300c monoclonal antibody, serially diluted 3-fold starting from 10 μg / ml, for 30 minutes in a CO2 incubator and washed twice with FACS buffer. FITC-conjugated anti-human IgG (H+L) diluted 1:100 in FACS buffer was then incubated for 30 minutes in a CO2 incubator and washed twice with FACS buffer. FITC signals were measured using a Beckman Coulter CytoFLEX instrument, and MFI values ​​were calculated using the CytExpert program. Using the MFI values, a sigmoidal curve was plotted using the sigmaplot program to calculate the EC50 (the effective concentration of drug that causes 50% of the maximum response). The EC50 values ​​were 2.7 nM for 293T cells and 2.6 nM for THP-1 cells.

[0166] As shown in Figure 10, the sigmoidal curve of the FACS binding results showed that the anti-CD300c monoclonal antibody prepared in Example 1 bound to CD300c overexpressed on the surface of THP-1 and 293T cells with strong avidity, confirming that the anti-CD300c monoclonal antibody binds to CD300c antigen-specifically.

[0167] Experimental Example 2.3. Confirmation of the binding ability of anti-CD300c monoclonal antibody to the CD300c antigen (I): Binding ELISA CD300c antigen (250 μg / mL) was diluted to a concentration of 800 μg / mL in coating buffer solution (0.1 M sodium carbonate, pH 9.0) and 100 μL was added to a 96-well microplate and incubated overnight at 4°C. The next day, the plate was washed three times with 200 μL of PBST. Then, 200 μL of blocking buffer solution (5% skim milk) was added to each well and blocked at room temperature for 1 hour. The anti-CD300c monoclonal antibody, CL7, was diluted to 200 μg / mL in PBS and applied to Nanodrop (product name: NanoDrop One / One). c The concentration was confirmed using a Thermo Fisher Scientific (manufacturer: Thermo Fisher Scientific). CL7 was then diluted 4-fold from 10 μg / mL with PBS, and 100 μL of each was added and incubated at room temperature for 1 hour. After incubation, the plate was washed three times with 200 μL of PBST. 100 μL of the secondary antibody (conjugated anti-Fc IgG) was added and incubated at room temperature for 1 hour. 200 μL of PBST was then added and washed three times. Next, TMB and hydrogen peroxide were mixed at a 1:1 ratio, and 100 μL was added to each well and incubated at room temperature for 7–9 minutes. The color development was stopped by adding 50 μL of 1N sulfuric acid, and the binding affinity was measured at 450 nm using a microplate reader (product name: Varioskan LUX).

[0168] As a result, as shown in Figure 11, it was confirmed that the anti-CD300c monoclonal antibody bound to CD300c in a concentration-dependent manner, indicating that the anti-CD300c monoclonal antibody has excellent binding affinity and specificity for the antigen CD300c.

[0169] Experimental Example 2.4. Confirmation of the binding strength of anti-CD300c monoclonal antibody to CD300c antigen (II): Surface plasmon resonance (SPR) To confirm the binding affinity between the antigen CD300c and the anti-CD300c monoclonal antibody CL7, a surface plasmon resonance experiment was carried out.

[0170] To immobilize CD300c onto a CM5 chip, 5 μg / ml of CD300c was diluted in 10 mM acetate buffer (pH 5.5). The flow rate was then set to 10 ml / min, with a target RU of 300 RU for each. Activation was performed with a mixture of 0.2 M EDC and 0.05 M NHS, followed by blocking with 1 M ethanolamine, resulting in immobilization of CD300c to a final RU of 399.2 RU. CL7 was then diluted in PBSP to concentrations of 0, 0.195, 0.39, 0.78, 1.56, 3.125, and 6.25 μg / ml, respectively, and a kinetics / affinity assay was performed with an association time of 240 seconds, a dissociation time of 900 seconds, and a flow rate of 30 μl / min. The surface was then regenerated with 50 mM NaOH at 30 μl / min for 30 seconds.

[0171] As a result, as shown in Figure 12, the KD value was analyzed to be 5.199E-10M, and the binding affinity of the anti-CD300c monoclonal antibody was confirmed to be 0.52nM, which is at the subnanomolar level, indicating the high binding strength of the anti-CD300c monoclonal antibody to the antigen.

[0172] Experimental Example 2.5. Confirmation of binding specificity of anti-CD300c monoclonal antibody to CD300c antigen (I) To confirm that the anti-CD300c monoclonal antibody CL7 specifically binds to CD300c and not to other B7 family proteins, we performed a binding ELISA. Specifically, ELISA plates were coated with CD300c antigen, CD300a antigen, or seven B7 family protein antigens (PD-L1 [B7-H1] (Sino Biological), ICOS Ligand [B7-H2] (Sino Biological), CD276 [B7-H3] (Sino Biological), B7-H4 (Sino Biological), CD80 [B7-1] (Sino Biological), CD86 [B7-2] (Sino Biological), and CD273 [PD-L2] (Sino Biological)) in a coating buffer solution (0.1 M sodium carbonate, pH 9.0) at a concentration of 8 μg / mL per well. The plates were then incubated overnight at 2°C–8°C to allow binding of the antigens to the plates. After washing three times with PBST to completely remove unbound antigen, 300 ml of blocking buffer (5% nonfat milk in PBST) was added to each well. Blocking was performed at room temperature for 1 hour, followed by washing again with PBST. CL7 was then diluted 4-fold in PBS and incubated at room temperature for 1 hour to bind to the antigen. After 1 hour, the wells were washed three times with PBST. Secondary antibody (HRP-conjugated anti-Fc IgG) diluted to 4 μg / mL in blocking buffer was then added and incubated at room temperature for another 1 hour. Unbound detection antibody was then removed with PBST, and TMB solution was added and incubated for 10 minutes to develop color. The color development reaction was terminated by adding 2N sulfuric acid solution, and the absorbance was measured at 450 nm to confirm the antibody specifically binding to the CD300c antigen.

[0173] As a result, as shown in FIG. 13, it was confirmed that the anti-CD300c monoclonal antibody did not bind to other similar proteins, but specifically recognized only CD300c.

[0174] Experimental Example 2.6. Confirmation of binding specificity of anti-CD300c monoclonal antibody to CD300c antigen (II) To confirm the specificity of the anti-CD300c monoclonal antibody CL7 for the CD300c antigen, we further investigated whether CL7 not only antagonizes the CD300c antigen, but also cross-reacts with the CD300a antigen, which has a similar protein sequence. More specifically, CD300a antigen (obtained from Sino Biological) was treated at concentrations of 0.039, 0.63, and 10 μg / mL, and then binding ELISA was performed using the same method as in Experimental Example 2.1.

[0175] As a result, as shown in FIG. 14, it was confirmed that the anti-CD300c monoclonal antibody did not bind to any antigens other than CD300c, and therefore exhibited high binding specificity only to the CD300c antigen.

[0176] Experimental Example 2.7. Comparison of overall survival time of various cancer patients according to CD300c expression level We compared overall survival according to CD300c expression levels in patients with kidney cancer (530 patients), pancreatic cancer (177 patients), and liver cancer (370 patients) obtained from the US TCGA (The Cancer Genome Atlas) database. Patients with each cancer were first classified according to their CD300c expression level. This level was determined by comparing the mean CD300c expression level by cancer type. Among kidney cancer patients, 394 had low CD300c expression levels, while 136 had high CD300c expression levels. Among pancreatic cancer patients, 57 had low CD300c expression levels, while 120 had high CD300c expression levels. Meanwhile, among liver cancer patients, 192 had low CD300c expression levels, while 178 had high CD300c expression levels. Overall survival according to CD300c expression level for each cancer type was analyzed using the Kaplan-Meier method. Next, the survival time of patients with high and low CD300c expression levels was compared using the log-rank test.

[0177] As a result, as shown in Figure 15, it was confirmed that patients with high CD300c expression levels had shorter survival times than patients with low CD300c expression levels, which is a significant result when considering the P value. These results not only indicate that CD300c expression is highly correlated with the survival time of cancer patients, but also that inhibiting CD300c expression or activity can be expected to have a therapeutic effect on cancer or extend survival time.

[0178] III. Anti-cancer effects of anti-CD300c monoclonal antibodies Experimental Example 3. Confirmation of anti-cancer effect by administration of anti-CD300c monoclonal antibody Experimental Example 3.1. Confirmation of T cell activation effect To confirm whether the anti-CD300c monoclonal antibody prepared in Example 1 exerts an anti-cancer effect through T cell activation, the amount of IL-2 (Interleukin-2) produced in human T cells following treatment with the anti-CD300c monoclonal antibody was measured. IL-2 is an immune factor that promotes the growth, proliferation, and differentiation of T cells. An increase in IL-2 production indicates increased stimulation of T cell differentiation, proliferation, and growth, thereby activating T cells. Specifically, anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody were added to a 96-well plate at a concentration of 2 μg / well each and allowed to set for 24 hours. After incubation at 1 x 10 5 Jurkat T cells (human T lymphocyte cell line) were treated with 10 μg / well of anti-CD300c monoclonal antibody. IL-2 production was measured using an ELISA kit (IL-2 Quantikine kit, R&D Systems) and compared with a control group not treated with anti-CD300c monoclonal antibody. The results are shown in Figure 16.

[0179] As shown in Figure 16, when Jurkat T cells activated by treatment with anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody were treated with anti-CD300c monoclonal antibody, the amount of IL-2 produced increased. These results confirmed that anti-CD300c monoclonal antibody activates T cells, thereby inducing anti-cancer immune responses and suppressing the growth of cancer tissue.

[0180] Experimental Example 3.2. Confirmation of promotion of differentiation into M1 macrophages (I): Measurement of the amount of macrophage differentiation marker (TNF-α) produced To confirm whether the anti-CD300c monoclonal antibodies selected in Example 1 can promote the differentiation of monocytes into M1 macrophages, 1.5x10 4 THP-1 (human monocytic cell line) cells were dispensed at 100 μg / mL per well and treated with 10 μg / mL anti-CD300c monoclonal antibody and / or 100 ng / mL LPS. After 48 hours of incubation, the production of TNF-α (Tumor necrosis factor-α), a differentiation marker for M1 macrophages, was measured using an ELISA kit (Human TNF-α Quantikine kit, R&D Systems). The results are shown in Figures 17 and 18.

[0181] As shown in Figure 17, the anti-CD300c monoclonal antibodies CL4, CL7, CL10, and SL18 increased TNF-α production by approximately two-fold or more compared to the control group (Con) treated with LPS alone.

[0182] Furthermore, as shown in Figure 18, compared to the control group (Con) treated with LPS alone, the experimental groups treated with anti-CD300c monoclonal antibody alone without LPS treatment showed increased TNF-α production compared to the control group.

[0183] Experimental Example 3.3. Confirmation of antibody concentration-dependent increase in differentiation potential into M1 macrophages To confirm that the anti-CD300c monoclonal antibody's induction of M1 macrophage differentiation increases depending on the concentration, the amount of TNF-α produced was measured using the same method as in Experimental Example 3.2. Cells were treated with anti-CD300c monoclonal antibody at concentrations of 10, 1, and 0.1 μg / mL. The results are shown in Figure 19. As shown in Figure 19, it was confirmed that the amount of TNF-α produced increased with increasing treatment concentration of anti-CD300c monoclonal antibody (CL7, CL10, or SL18).

[0184] To confirm the concentration in more detail, anti-CD300c monoclonal antibody (CL7) was added at concentrations of 10, 5, 2.5, 1.25, 0.625, 0.313, 0.157, and 0.079 μg / mL, and the amount of TNF-α produced was confirmed. The results are shown in Figure 20. As shown in Figure 20, it was confirmed that the amount of TNF-α produced increased as the concentration of anti-CD300c monoclonal antibody used increased.

[0185] Experimental Example 3.4. Confirmation of promotion of differentiation into M1 macrophages (II): Cell morphology observation To confirm the differentiation of monocytes into M1 macrophages by cell morphology, THP-1 cells were treated with 10 μg / ml of anti-CD300c monoclonal antibody and cultured for 48 hours, after which the cell morphology was observed under a microscope. The results are shown in Figure 21.

[0186] As shown in Figure 21, in the experimental group (CL7) treated with anti-CD300c monoclonal antibody, the morphology of THP-1 cells changed from suspension cells to round, adherent cells, which are the morphology of M1 macrophages. These results confirmed that treatment with anti-CD300c monoclonal antibody promotes the differentiation of monocytes into M1 macrophages.

[0187] Experimental Example 3.5. Reconfirmation of promotion of differentiation into M1 macrophages To confirm whether CL7 anti-CD300c monoclonal antibody promotes the differentiation of human monocytes into M1 macrophages, the secretion levels of TNF-α, IL-1β (Interleukin-1β), and IL-8 (Interleukin-8) were measured using ELISA kits. 4 THP-1 cells were dispensed at 100 cells / well and treated with 10 μg / mL of anti-CD300c monoclonal antibody. After 48 hours of incubation, the production of M1 macrophage differentiation markers TNF-α, IL-1β, and IL-8 was measured using an ELISA kit (Human TNF-α Quantikine kit, R&D Systems). The results are shown in Figure 22.

[0188] As shown in Figure 22, all three M1 macrophage differentiation markers were found to be increased in the experimental group (CL7) treated with anti-CD300c monoclonal antibody compared to the control group (Con) not treated with anti-CD300c monoclonal antibody.

[0189] Experimental Example 3.6. Confirmation of the redifferentiation ability of M2 macrophages into M1 macrophages To determine whether anti-CD300c monoclonal antibody can redifferentiate M2 macrophages into M1 macrophages, 1.5x10 cells were cultured in a 96-well plate. 4 THP-1 cells were dispensed at 1000 cells / well and pretreated with 320 nM PMA for 6 hours. After pretreatment, the cells were treated with 20 ng / mL IL-4 (Interleukin-4) and IL-13 (Interleukin-13) and 10 μg / mL anti-CD300c monoclonal antibody for 18 hours. The production of TNF-α, IL-1β, and IL-8 was measured using ELISA kits. The results are shown in Figures 23-25.

[0190] As shown in Figures 23 to 25, without PMA pretreatment, the levels of TNF-α, IL-1β, and IL-8 produced increased in the experimental group treated with IL-4, IL-13, and anti-CD300c monoclonal antibody, and similarly, with PMA pretreatment, the levels of TNF-α, IL-1β, and IL-8 produced increased in the experimental group treated with IL-4, IL-13, and anti-CD300c monoclonal antibody. These results confirm that anti-CD300c monoclonal antibody effectively redifferentiates M2 macrophages back into M1 macrophages.

[0191] Experimental Example 3.7. Confirmation of differentiation and redifferentiation ability into M1 macrophages To confirm the differentiation and redifferentiation ability of anti-CD300c monoclonal antibody to M1 macrophages, 1.5x10 cells were cultured in a 96-well plate. 4 THP-1 cells were dispensed at 100 μg / mL per well and pretreated with 10 μg / mL anti-CD300c monoclonal antibody for 48 hours. Then, cells were treated with 100 ng / mL PMA, 100 ng / mL LPS, and 20 ng / mL IL-4 and IL-13 for 24 hours. TNF-α production was measured using an ELISA kit. The results are shown in Figure 26.

[0192] As shown in Figure 26, compared with the M0 macrophage control group treated with PMA alone, the M1 macrophage control group treated with LPS alone, and the M2 macrophage control group treated with IL-4 and IL-13 alone, the experimental group pretreated with anti-CD300c monoclonal antibody showed significantly increased TNF-α production. These results confirmed that anti-CD300c monoclonal antibody has excellent abilities to differentiate M0 macrophages into M1 macrophages, differentiate THP-1 macrophages into M1 macrophages, and redifferentiate M2 macrophages into M1 macrophages.

[0193] Experimental Example 4. Confirmation of interspecies cross-reactivity of anti-CD300c monoclonal antibody by observing anti-cancer effect Experimental Example 4.1. Confirmation of the growth inhibitory effect on human cancer cells To confirm the effect of monoclonal antibodies targeting CD300c on cancer cell growth, cell proliferation assays were performed using A549 (human lung cancer cell line). More specifically, 2 x 10 cells were cultured in a 96-well plate in the presence of 0% fetal bovine serum (FBS). 4 Cells were aliquoted and plated at 6 x 10 cells per well in 0.1% fetal bovine serum. 3 The cells were then aliquoted and treated with 10 μg / mL of anti-CD300c monoclonal antibody and cultured for 5 days. The tumor cell growth inhibitory effect of anti-CD300c monoclonal antibody was confirmed by treating with CCK-8 (DOJINDO) and measuring absorbance at OD 450 nm. The results are shown in Figures 27 and 28.

[0194] As shown in FIG. 27, all the cells except for SK11 and SK17 were found to have the effect of inhibiting the proliferation of cancer cells under the condition of 0% FBS.

[0195] As shown in FIG. 28, it was confirmed that under the condition of 0.1% FBS, all of the anti-CD300c monoclonal antibodies used in the experiment were effective in suppressing the proliferation of cancer cells.

[0196] Experimental Example 4.2. Confirmation of the cancer cell growth inhibitory effect at different concentrations of anti-CD300c monoclonal antibody To confirm the inhibitory effect of anti-CD300c monoclonal antibody on cancer cell growth depending on the concentration, 2 x 10 4 A549 cells were aliquoted, treated with 10 μg / mL of anti-CD300c monoclonal antibody, and cultured for 5 days. After treatment with CCK-8 (DOJINDO) for 3 hours, the absorbance was measured at OD 450 nm to confirm the inhibitory effect of anti-CD300c monoclonal antibody on cancer cell growth. The results are shown in Figure 29.

[0197] As shown in FIG. 29, it was confirmed that the growth of cancer cells was inhibited as the concentration of anti-CD300c monoclonal antibody increased.

[0198] Experimental Example 4.3. Confirmation of increased differentiation potential into M1 macrophages in mice To confirm whether anti-CD300c monoclonal antibody can promote differentiation of mouse macrophages into M1 macrophages, mouse macrophages (Raw264.7) were cultured at 1x10 in a 96-well plate. 4 After dispensing at a concentration of 100 cells / well, the cells were treated with 10 μg / mL of anti-CD300c monoclonal antibody and cultured. The amount of TNF-α produced was determined using an ELISA kit. The results are shown in Figure 30.

[0199] As shown in Figure 30, the experimental group treated with anti-CD300c monoclonal antibody showed increased TNF-α production. These results demonstrate that anti-CD300c monoclonal antibody has cross-reactivity that promotes differentiation into M1 macrophages in a similar manner in both humans and mice.

[0200] Experimental Example 4.4. Confirmation of the growth inhibitory effect on mouse cancer cells To confirm whether the anti-CD300c monoclonal antibodies CL7, CL10, and SL18 exerted anti-cancer effects, CT26 (a mouse colon cancer cell line) was cultured in a 96-well plate at 1x10 4 The cells were dispensed at a concentration of 10 μg / well, treated with 10 μg / mL of monoclonal antibody, and cultured for 5 days, after which cell proliferation analysis was performed using CCK-8 detection.

[0201] As shown in Figure 31, the anti-CD300c monoclonal antibody exhibited 66% (CL7), 15% (CL10), and 38% (SL18) cancer cell proliferation inhibitory effects compared to the control group, respectively, demonstrating its therapeutic effect in cancer treatment in mice. This demonstrates that the anti-CD300c monoclonal antibody has cross-reactivity, functioning similarly in both humans and mice to exert its anti-cancer effect.

[0202] Experimental Example 5. Comparison of in vitro anti-cancer effects between anti-CD300c monoclonal antibody and existing immunosuppressants The sources of the immunological anticancer drugs used in the following experimental examples are as follows: Imfinzi (AstraZeneca) and Keytruda (Merck Sharp & Dohme).

[0203] Experimental Example 5.1. Comparison of the differentiation ability into M1 macrophages between anti-CD300c monoclonal antibody and existing immunosuppressive agents: Measurement of the production of three differentiation markers (TNF-α, IL-1β, and IL-8) For comparison, the amount of TNF-α produced was measured using an ELISA kit in the same manner as in Experimental Example 3.2. Imfinzi, an existing immunosuppressant, was used at a concentration of 10 μg / mL. The results are shown in Figure 32.

[0204] As shown in Figure 32, anti-CD300c monoclonal antibody significantly increased TNF-α production compared to the control group treated with Imfinzi (Imf) alone. These results confirmed that anti-CD300c monoclonal antibody significantly increased M1 macrophage differentiation compared to known immunosuppressive anticancer agents.

[0205] For comparison with other immunotherapy drugs, cells were treated with the anti-PD-L1 immunotherapy drug Imfinzi, the anti-PD-1 immunotherapy drug Keytruda, and an isotype control (immunoglobulin G) antibody at a concentration of 10 μg / mL, and the production of TNF-α, IL-1β, and IL-8 was measured using ELISA kits. The results are shown in Figures 33 to 35.

[0206] As shown in Figures 33 to 35, it was confirmed that the anti-CD300c monoclonal antibody significantly increased the production of TNF-α, IL-1β, and IL-8 compared to Imfinzi, Keytruda, and IgG antibodies. These results confirmed that the anti-CD300c monoclonal antibody significantly increased the promotion of differentiation into M1 macrophages compared to existing immunosuppressive anticancer drugs.

[0207] Experimental Example 5.2. Comparison of the differentiation ability of M0 macrophages to M1 macrophages between anti-CD300c monoclonal antibody and existing immunosuppressive agents To compare the differentiation ability of anti-CD300c monoclonal antibody and immunosuppressants from M0 to M1 macrophages, 1.5x10 cells were cultured in a 96-well plate. 4 THP-1 cells were dispensed at 100 cells / well and treated with 10 μg / mL anti-CD300c monoclonal antibody, 10 μg / mL Imfinzi, and / or 200 nM PMA (phorbol-12-myristate-13-acetate). After 48 hours of incubation, TNF-α production was measured using an ELISA kit. The results are shown in Figure 36.

[0208] As shown in Figure 36, the control group treated with the immunosuppressant Imfinzi alone did not produce TNF-α, while the experimental group treated with anti-CD300c monoclonal antibody alone showed increased TNF-α production. Furthermore, when THP-1 cells were treated with PMA to differentiate into M0 macrophages, the experimental group treated with anti-CD300c monoclonal antibody showed significantly higher TNF-α production than the experimental group treated with Imfinzi. These results confirm that anti-CD300c monoclonal antibody promotes the differentiation of M0 macrophages into M1 macrophages compared to existing immunosuppressant drugs.

[0209] Experimental Example 5.3. Comparison of M1 macrophage differentiation ability between anti-CD300c monoclonal antibody and existing immunosuppressive agents To compare the differentiation ability of anti-CD300c monoclonal antibody and existing immunological anticancer agents into M1 macrophages, the amount of TNF-α produced was determined using the same method as in Experimental Example 3.2. The results are shown in Figure 37.

[0210] As shown in Figure 37, when monocytes were differentiated into M1 macrophages by treatment with LPS, there was no significant difference in the amount of TNF-α produced in the experimental group treated with both Imfinzi and LPS, but it was confirmed that the amount of TNF-α produced in the experimental group treated with both anti-CD300c monoclonal antibody and LPS was significantly increased compared to the experimental group treated with anti-CD300c monoclonal antibody alone.

[0211] Experimental Example 5.4. Comparison of the cancer cell growth inhibitory effects between anti-CD300c monoclonal antibody and existing immunosuppressive agents To compare the cancer cell growth inhibitory effects of anti-CD300c monoclonal antibodies with existing immunological anticancer drugs, we examined the cell growth inhibitory effects using A549 (human lung cancer cell line) and MDA-MB-231 (human breast cancer cell line). More specifically, 2 x 10 cells were cultured in a 96-well plate with 0% fetal bovine serum (FBS). 4 Cells were aliquoted and plated at 6 x 10 cells per well in 0.1% fetal bovine serum. 3 The cells were then aliquoted, treated with 10 μg / mL of anti-CD300c monoclonal antibody, and cultured for 5 days before being observed under an optical microscope. The results are shown in Figures 38 and 39.

[0212] As shown in Figure 38, it was confirmed that the anti-CD300c monoclonal antibody inhibited the proliferation of cancer cells more effectively than the immunosuppressant Imfinzi in the A549 cell line.

[0213] As shown in Figure 39, it was confirmed that the anti-CD300c monoclonal antibody inhibited cancer cell proliferation more effectively than the immunosuppressant Imfinzi in the MDA-MB-231 cell line.

[0214] Experimental Example 6: Confirmation of in vivo anti-cancer effect of anti-CD300c monoclonal antibody Experimental Example 6.1. Confirmation of Increase in Tumor-Associated Macrophages (TAM) To confirm the effect of the anti-CD300c monoclonal antibody CL7 on tumor-associated macrophages in vivo, 2x10 colon cancer cell lines (CT26) were used. 5 The cells were subcutaneously transplanted into 8-week-old BALB / c mice to create a syngeneic mouse tumor model. All animal care and experiments were carried out in a specific pathogen-free (SPF) facility. After 12 days of transplantation, the tumors grew to 50-100 mm. 3 Mice were administered anti-CD300c monoclonal antibodies (mAbs) and the control group received an equal volume of phosphate buffered saline (PBS). Mice were intraperitoneally injected at a dose of 25 mg / kg twice weekly for two weeks for a total of four injections. Twenty-five days after injection, mice were sacrificed and tumor tissue was collected from six mice in each of the CL7 25 mg / kg groups, which showed the highest antitumor effect compared to the control group. Tumor tissue was then excised and incubated in a mixture of collagenase D (20 mg / ml) and DNase I (2 mg / ml) at 37°C for 1 hour. It was then filtered through a 70 μm cell strainer to lyse red blood cells and refiltered through a nylon mesh. The single-cell suspension was then blocked with CD16 / 32 antibody (obtained from Invitrogen), and cells were stained with a cell viability stain and antibodies against F4 / 80, a marker for total macrophages, and iNOS, a marker for M1 macrophages (obtained from Abcam). Data were then read on a CytoFLEX flow cytometer and analyzed using FlowJo software.

[0215] As a result, as shown in Figure 40, it was confirmed that the expression level of M1 tumor-associated macrophages in mouse cancer tissue increased when anti-CD300c monoclonal antibody was administered alone, which means that administration of anti-CD300c monoclonal antibody increases tumor-associated macrophages in cancer tissue, suppressing cancer growth.

[0216] Experimental Example 6.2. Confirmation of increase in cytotoxic T cells To confirm the effect of the anti-CD300c monoclonal antibody CL7 on CD8+ T cells in vivo, an allograft mouse tumor model was prepared as described in Experimental Example 6.1 and administered at the same dose. Twenty-five days after injection, the mice were sacrificed and tumor tissue was collected from six mice in each of the 25 mg / kg CL7 treatment groups, which showed the highest antitumor effect compared to the control group. The tumor tissue was then excised and incubated at 37°C for 1 hour in a mixture of collagenase D (20 mg / ml) and DNase I (2 mg / ml). It was then filtered through a 70 μm cell strainer to lyse red blood cells and refiltered through a nylon mesh. The single-cell suspension was then blocked with CD16 / 32 (Invitrogen) antibodies and stained with cell viability staining solution, CD8+ antibodies (Abcam), and CD4+ antibodies (Abcam). Data were then read on a CytoFLEX flow cytometer and analyzed with FlowJo software.

[0217] As a result, as shown in Figure 41, it was confirmed that treatment with anti-CD300c monoclonal antibody alone increased the number of intratumoral CD8+ T cells, indicating that administration of anti-CD300c monoclonal antibody increases intratumoral cytotoxic T cells, resulting in a cancer therapeutic effect.

[0218] Experimental Example 6.3. Confirmation of tumor-specific increase in cytotoxic T cells To confirm whether the anti-CD300c monoclonal antibody CL7 increases CD8+ T cell numbers in a tumor-specific manner, an allograft mouse tumor model was prepared as described in Experimental Example 6.1 and administered at the same dose. Twenty-five days after injection, the mice were sacrificed and tumor tissue was collected from six mice in each of the CL7 25 mg / kg groups, which showed the highest antitumor effect compared to the control group. The tumor tissue was then excised and incubated at 37°C for 1 hour in a mixture of collagenase D (20 mg / ml) and DNase I (2 mg / ml). It was then filtered through a 70 μm cell strainer, red blood cells were lysed, and the tissue was refiltered through a nylon mesh. The single-cell suspension was then blocked with CD16 / 32 antibody (obtained from Invitrogen), and the cells were stained with cell viability stain and AH1 tetramer antibody (obtained from Abcam). Data were then read using a CytoFLEX flow cytometer and analyzed using FlowJo software.

[0219] As a result, as shown in Figure 42, when anti-CD300c monoclonal antibody was administered alone, the expression of AH1-tetramer, a CT26 tumor marker, increased in CD8+ T cells, and the number of CD8+ T cells increased in a tumor (CT26)-specific manner. This indicates that administration of anti-CD300c monoclonal antibody increased CD8+ T cells to target and suppress CT26 cancer cells.

[0220] Experimental Example 6.4. Confirmation of increased activity of cytotoxic T cells To confirm the effect of the anti-CD300c monoclonal antibody CL7 on CD8+ T cells in vivo, an allograft mouse tumor model was prepared as in Experimental Example 6.1 and administered at the same dose. The mice were sacrificed 25 days after injection, and spleens were collected from six mice in each of the 25 mg / kg CL7 groups, which showed the highest antitumor effect compared to the control group. IFN-γ was then measured using ELISPOT assay to confirm the results. Specifically, a Mouse IFN-γ ELISpot kit from R&D Systems (#EL485) was purchased and IFN-γ was measured according to the kit's protocol.

[0221] As a result, as shown in Figure 43, it was confirmed that treatment with anti-CD300C monoclonal antibody alone increased IFN-γ expression. This indicates that administration of anti-CD300C monoclonal antibody alone not only increased the number of CD8+ T cells (see Figure 41) but also increased the activity of CD8+ T cells, thereby suppressing cancer growth in multiple ways and achieving a cancer treatment effect.

[0222] Experimental Example 6.5. Confirmation of an increase in cytotoxic T cells compared to regulatory T cells To confirm the in vivo effect of the anti-CD300c monoclonal antibody CL7 on the increase of cytotoxic T cells relative to regulatory T cells, an allograft mouse tumor model was prepared as in Experimental Example 6.1 and administered at the same dose. Twenty-five days after injection, the mice were sacrificed and tumor tissue was collected from six mice in each of the 25 mg / kg CL7 treatment groups, which showed the highest antitumor effect compared to the control group. The tumor tissue was then excised and incubated in a mixture of collagenase D (20 mg / ml) and DNase I (2 mg / ml) at 37°C for 1 hour. It was then filtered through a 70 μm cell strainer, red blood cells were lysed, and the tissue was refiltered through a nylon mesh. The single-cell suspension was then blocked with CD16 / 32 (Invitrogen) antibody and stained with a cell viability stain, antibodies against the Treg marker proteins CD25 (Sino Biological) and Foxp3 (Abcam) (Sino Biological), CD3+ antibody, and CD8+ antibody. Data were then read using a CytoFLEX flow cytometer and analyzed using FlowJo software.

[0223] As a result, as shown in Figure 44, it was confirmed that CD8+ T cells were increased compared to Treg T cells when anti-CD300C monoclonal antibody was administered alone, which means that the CD8+ T cells, whose numbers increased with the administration of anti-CD300C monoclonal antibody, further suppressed cancer growth.

[0224] Experimental Example 6.6. Confirmation of effects on cytotoxic T cells, regulatory T cells, and tumor-associated macrophages To confirm the effect of anti-CD300c monoclonal antibody CL7 on cytotoxic T cells, regulatory T cells, and tumor-associated macrophages, the following experiment was conducted. An allograft mouse tumor model was prepared as in Experimental Example 6.1. Colon cancer cell lines were transplanted, and after 12 days, tumors grew to 50-100 mm. 3Mice were administered anti-CD300c monoclonal antibodies (mAbs) or phosphate buffered saline (PBS) in the control group. Mice were intraperitoneally injected at a dose of 25 mg / kg twice weekly for two weeks for a total of four injections. Twenty-five days after injection, the mice were sacrificed and tumor tissue was collected from six mice in each of the CL7 25 mg / kg groups, which showed the highest antitumor effect compared to the control group. The tumor tissue was then excised and incubated in a mixture of collagenase D (20 mg / ml) and DNase I (2 mg / ml) at 37°C for one hour. It was then filtered through a 70 μm cell strainer to lyse red blood cells and refiltered through a nylon mesh. The single-cell suspension was then blocked with CD16 / 32 antibody (obtained from Invitrogen) and stained with a cell viability stain and CD8+ and CD4+ antibodies, which are CD8+ T cell markers, or with Foxp3 and CD4+ antibodies, which are Treg cell markers, or with antibodies against F4 / 80, a marker for total macrophages, and iNOS, an M1 macrophage marker (obtained from Abcam). Data were then read using a CytoFLEX flow cytometer and analyzed using FlowJo software. The results are shown in Figure 45.

[0225] As shown in Figure 45, anti-CD300c monoclonal antibody (CL7) significantly increased activated CD8+ T cells, suppressed regulatory T cells, and repolarized tumor-associated macrophages toward the M1 phenotype.

[0226] Experimental Example 6.7. Confirmation of in vivo tumor growth inhibitory effect To confirm the anti-cancer effect of the anti-CD300c monoclonal antibody CL7 in vivo, 2x10 colon cancer cell lines (CT26) were cultured. 5 The cells were subcutaneously transplanted into 8-week-old BALB / c mice to create an allograft mouse tumor model. All animal care and experiments were carried out in an SPF facility. On day 11 (D11) after colon cancer cell transplantation, tumors grew to 50-100 mm. 3Mice were administered anti-CD300c monoclonal antibodies at doses of 1 mg / kg, 5 mg / kg, 10 mg / kg, or 25 mg / kg, respectively, and the control group received an equal volume of phosphate buffered saline (PBS). Specifically, mice were intraperitoneally injected with each dose twice a week for a total of four injections over two weeks (Days 11, 14, 18, and 21). Tumor volume was measured for 25 days. The results are shown in Figure 46.

[0227] As shown in FIG. 46, it was confirmed that the anti-CD300c monoclonal antibody CL7 delayed the growth of CT26 colon cancer cells in a dose-dependent manner.

[0228] IV. Changes in biomarker expression following administration of anti-CD300c monoclonal antibody Example 2. Changes in expression of immune cell-related markers and tumor microenvironment-related markers following administration of anti-CD300c monoclonal antibody Example 2.1. Nanostring Immune Profiling The anti-CD300c monoclonal antibody (CL7) prepared in Example 1 was administered to a solid tumor model. To confirm the changes in the expression of immune cells and tumor microenvironment-related markers, 2x10 colon cancer cell line (CT26) was used. 5 The cells were subcutaneously transplanted into 8-week-old BALB / c mice to create an allograft mouse tumor model. All animal care and experiments were carried out in an SPF facility. After 12 days of transplantation of the colon cancer cell line, tumors grew to 50-100 mm. 3Mice were administered anti-CD300c monoclonal antibodies, while the control group received an equal volume of phosphate buffered saline (PBS). Mice were injected intraperitoneally at a dose of 25 mg / kg twice a week for two weeks, for a total of four injections. Twenty-five days after injection, the mice were euthanized and tumor tissue was prepared. RNA was extracted and purified from the tumor tissue, and changes in dendritic cell markers, macrophage markers, tumor microenvironment (TME) markers, Th1 response markers, and Th2 response markers were confirmed using NanoString immunoprofiling.

[0229] The results of the NanoString immune profiling are shown in Figure 47. This confirmed that administration of anti-CD300c monoclonal antibody extensively reprogrammed the tumor immune microenvironment.

[0230] In addition, changes in dendritic cell markers, macrophage markers, tumor microenvironment markers, Th1 response markers, and Th2 response markers following administration of anti-CD300c monoclonal antibody compared to the control group were observed, and the results are shown in Figure 48. As shown in Figure 48, administration of anti-CD300c monoclonal antibody significantly increased the expression of dendritic cell markers Bst2, CCL8, and Xcl1; significantly increased the expression of M1 macrophage markers CCR7 and CD80; decreased the expression of vegfa, pdgfrb, Col4a1, and Hif1a, which support cancer growth in the tumor microenvironment; and increased the expression of Tbx21, Stat1, Stat4, Ifn-g, and Cxcr3, markers that can confirm Th1 response.

[0231] Example 2.2. Changes in the expression of immune barrier markers Based on the NanoString immune profiling results obtained in Example 2.1, we determined which immune barrier markers showed significant differences in expression when anti-CD300c monoclonal antibody was administered to an allograft mouse tumor model compared to the control group.

[0232] The results are shown in Figure 49. Administration of anti-CD300c monoclonal antibody increased the expression of PD-1, CTLA-4, and Lag3 in the inhibitory immune barrier (inhibitory IC), and also increased the expression of ICOS, OX40, Gitr, Cd27, and Cd28 in the agonistic immune barrier (agonistic IC).

[0233] These results are of considerable significance in that they may provide useful information regarding which immune barrier immune anticancer agent should be selected when administering an anti-CD300c monoclonal antibody in combination with an additional immune anticancer agent to achieve further improved anticancer efficacy.

[0234] V. Combination of anti-CD300c monoclonal antibody and immunotherapy Example 3. Combined administration of anti-CD300c monoclonal antibody (CL7) and immunosuppressant The anti-CD300c monoclonal antibody (CL7) prepared in Example 1 was used in combination with other immunosuppressive anticancer agents, such as the anti-PD-L1 antibodies Imfinzi® and Opdivo®, the anti-PD-1 antibody Keytruda, the anti-CD47 antibody (αCD47), and the anti-CTLA-4 antibody, and the results were observed.

[0235] The sources of these immune anti-cancer drugs are as follows: Imfinzi (AstraZeneca); Opdivo, an anti-CTLA-4 antibody (Bristol Myers Squibb Company); Keytruda (Merck Sharp & Dohme); and anti-CD47 antibody (Abcam).

[0236] Experimental Example 7. Confirmation of (synergistic) increase in macrophage activity by combined use Experimental Example 7.1. Confirmation of increase in M1 macrophages To confirm the differentiation into M1 macrophages by cell morphology when monocytes were treated with the anti-CD300c monoclonal antibody CL7 prepared in Example 1 in combination with immunological anticancer agents such as the anti-PD-L1 antibody Imfinzi, the anti-PD-1 antibody Keytruda, and the anti-CD47 antibody (αCD47), THP-1 (human monocyte cell line) was treated with 10 μg / ml of each of the anti-CD300c monoclonal antibody and immunological anticancer agent, either alone or in combination, and cultured for 48 hours, after which the cell morphology was observed under a microscope.

[0237] As a result, as shown in Figure 50, when THP-1 cells were treated with an anti-CD300c monoclonal antibody in combination with an immunosuppressant, the morphology of the cells changed from suspension cells to round, adherent cells, which are the morphology of M1 macrophages, compared to when the cells were treated with an immunosuppressant alone. These results confirmed that the combination of an anti-CD300c monoclonal antibody and an immunosuppressant further promoted the differentiation of monocytes into M1 macrophages.

[0238] Experimental Example 7.2. Confirmation of increase in M1 macrophage markers To confirm whether the induction of monocyte differentiation into M1 macrophages was increased when the anti-CD300c monoclonal antibody CL7 was used in combination with the anti-PD-L1 antibody Imfinzi, the anti-PD-1 antibody Opdivo, the anti-PD-1 antibody Keytruda, the anti-CD47 antibody, and the anti-CTLA-4 antibody, 1.5x10 cells were cultured in a 96-well plate. 4 THP-1 cells were dispensed into wells and treated with anti-CD300c monoclonal antibody and anticancer drugs at 10 μg / mL, either alone or in combination. After 48 hours of incubation, the production of M1 macrophage differentiation markers TNF-α (Tumor necrosis factor-α), IL-1b, and IL-8 was measured using an ELISA kit (Human TNF-α Quantikine kit, R&D Systems).

[0239] As a result, as shown in Figure 51, treatment with anti-CD300c monoclonal antibody alone increased the production of all three differentiation markers, with IL-8 production being particularly notable. Furthermore, as shown in Figure 52, treatment with anti-CD300c monoclonal antibody in combination with Imfinzi, Opdivo, Keytruda, and αCD47 further increased the production of TNF-α, a marker for M1 macrophages, compared to treatment with anti-CD300c monoclonal antibody alone. This indicates that treatment with anti-CD300c monoclonal antibody in combination with anti-PD-1 antibody and / or anti-CD47 antibody results in greater differentiation of monocytes into M1 macrophages than treatment with anti-CD300c monoclonal antibody alone.

[0240] Experimental Example 7.3. Confirmation of reduction in M2 macrophage markers To confirm whether the induction of monocyte differentiation into M2 macrophages was reduced when anti-CD300c monoclonal antibody was administered in combination with immunotherapy drugs such as Imfinzi, Opdivo, Keytruda, anti-CTLA-4, or αCD47, 1.5x10 cells were cultured in a 96-well plate. 4 THP-1 cells / well were dispensed and pretreated with 320 nM PMA for 6 hours. After pretreatment, cells were treated with 20 ng / mL IL-4 (Interleukin-4) and IL-13 (Interleukin-13), along with 10 μg / mL anti-CD300c monoclonal antibody and anticancer drugs, either alone or in combination, for 48 hours. The production of IL-10 and IL-12, which are differentiation markers for M2 macrophages, was then measured using an ELISA kit (R&D Systems).

[0241] As a result, it was confirmed that the production of IL-10 and IL-12 was reduced by more than 30% when treated in combination with Imfinzi, Opdivo, Keytruda, and αCD47 compared to treatment with anti-CD300c monoclonal antibody alone.

[0242] Experimental Example 7.4. Confirmation of increased M1 macrophage differentiation potential To confirm that the anti-CD300c monoclonal antibody CL7, in combination with immunosuppressive agents such as anti-PD-1, anti-PD-L1, anti-CTLA-4, and anti-CD47 antibodies, enhances the differentiation of monocytes into M1 macrophages, we examined the signal transduction of MAPK (mitogen-activated protein kinase), IkB, and NF-kB, which are representative signals for M1 macrophage differentiation. 5 THP-1 cells / well were dispensed and treated with 10 μg / mL anti-CD300c monoclonal antibody, 10 μg / mL Imfinzi, and / or 10 μg / mL Keytruda. Controls were treated with the same volume of phosphate buffered saline (PBS). After 48 hours of culture, phosphorylated SAPK / JNK, phosphorylated ERK, and phosphorylated p38 were detected in MAPK signaling, phosphorylated NF-kB in NF-kB signaling, and phosphorylated IkB in IkB signaling by Western blotting. The results are shown in Figures 53 to 55.

[0243] Figures 53, 54, and 55 show the results of investigating MAPK, NF-kB, and IkB signaling, respectively. It was confirmed that the levels of phosphorylated MAPK, IkB, and NF-kB increased when anti-CD300c was treated in combination with immunological anticancer agents such as anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, and anti-CD47 antibody compared to treatment with anti-CD300c monoclonal antibody alone. This confirms that cell signaling leading to M1 macrophage differentiation is enhanced when anti-CD300c monoclonal antibody is treated in combination with immunological anticancer agents such as anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, and anti-CD47 antibody compared to treatment with anti-CD300c monoclonal antibody alone.

[0244] Experimental Example 8. Confirmation of (synergistic) increase in cancer cell growth inhibitory effect by combined use (in vitro) Experimental Example 8.1. Confirmation of cell suicide signal We investigated whether the apoptosis signaling was increased when CL7, an anti-CD300c monoclonal antibody, was used in combination with immunotherapy drugs such as anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, and anti-CD47 antibody. 5 Cells were dispensed into A549 plates at 10 μg / mL and treated with 10 μg / mL of anti-CD300c monoclonal antibody, 10 μg / mL of Imfinzi, Keytruda, Opdivo, or anti-CD47 antibody, either alone or in combination. After 48 hours of culture, apoptosis signaling and cell cycle signaling were examined by Western blotting. Markers of apoptosis signaling were identified, including cleaved caspase-9, caspase-3, caspase-2, and caspase-8, and cell cycle signaling markers were identified, including cyclin D1, CDK2, p27kip1, CDK6, cyclin D3, p21 Waf1, and Cip1.

[0245] As shown in Figure 56, apoptosis signals were increased when anti-CD300c monoclonal antibody was combined with Imfinzi, an anti-PD-1 antibody, compared to treatment alone, and the levels of cleaved-caspase 9 and p21 increased and cyclin D1 decreased when anti-CD300c monoclonal antibody was combined with immunological anticancer agents such as anti-PD1, anti-PD-L1, anti-CTLA-4, and anti-CD47. These results confirm that apoptosis of cancer cells is more effectively induced when anti-CD300c monoclonal antibody is combined with immunological anticancer agents such as anti-PD-1, anti-PD-L1, anti-CTLA-4, and anti-CD47 than when treated alone.

[0246] Experimental Example 8.2. Confirmation of the growth inhibitory effect on cancer cell lines To confirm the cancer cell growth inhibitory effect of the combined administration of anti-CD300c monoclonal antibody CL7 and immunotherapy, we compared the cancer cell growth inhibitory effect using A549 (human lung cancer cell line) and MDA-MB-231 (human breast cancer cell line). Specifically, in the absence of fetal bovine serum (FBS) in a 96-well plate, 2 x 104 Cells (A549) or 3x10 4 MDA-MB-231 cells were aliquoted and cultured at a density of 6 x 10 cells per well in 0.1% fetal bovine serum. 3 Cells (A549) or 1x10 4 MDA-MB-231 cells were then aliquoted and treated with 10 μg / mL of anti-CD300c monoclonal antibody and Imfinzi, either alone or in combination, and cultured for 5 days. As a control, cells were treated with the same volume of phosphate buffered saline (PBS). CCK-8 (DOJINDO) was then added, and the absorbance was measured at OD 450 nm. The results are shown in Figure 57 (A549) and Figure 58 (MDA-MB-231).

[0247] When treated with the A549 cell line, as shown in Figure 57, in the absence of FBS, when anti-CD300c monoclonal antibody was treated alone, the cell growth inhibitory effect was 17% higher than the control group, and when treated in combination with Imfinzi, the effect was 34% higher.

[0248] When MDA-MB-231 cells were treated with 0.1% FBS, as shown in Figure 58, treatment with anti-CD300c monoclonal antibody alone resulted in a 19% greater inhibitory effect on cancer cell growth compared to the control group. Treatment with anti-CD300c monoclonal antibody and anti-CD47 antibody in combination resulted in a 45% greater inhibitory effect, and treatment with anti-CD300c monoclonal antibody, anti-CD47 antibody, and Imfinzi resulted in a 51% greater inhibitory effect. Under 0.1% FBS, treatment with anti-CD300c monoclonal antibody alone resulted in a 19% greater inhibitory effect on cancer cell growth compared to the control group. Treatment with anti-CD300c monoclonal antibody and anti-CD47 antibody in combination resulted in a 22% greater inhibitory effect, and treatment with anti-CD300c monoclonal antibody, anti-CD47 antibody, and Imfinzi resulted in a 32% greater inhibitory effect.

[0249] These results confirmed that cancer cell growth was more effectively suppressed when anti-CD300c monoclonal antibody was used in combination with immunosuppressive anti-cancer agents such as anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, and anti-CD47 antibody than when it was used alone.

[0250] Experimental Example 9: Confirmation of (synergistic) increase in in vivo anti-cancer effect by combined use (colon cancer mouse model) Experimental Example 9.1. Confirmation of in vivo tumor growth inhibitory effect To confirm the anti-cancer effect of the anti-CD300c monoclonal antibody CL7 in vivo, 2x10 colon cancer cell lines (CT26) were cultured. 5 The cells were subcutaneously transplanted into 8-week-old BALB / c mice to create an allograft mouse tumor model. All animal care and experiments were carried out in an SPF facility. On day 12 (D12) after colon cancer cell transplantation, tumors grew to 50-100 mm. 3 Mice were administered anti-CD300c monoclonal antibody and anti-PD-1 antibody purchased from BioXcell, either alone or in combination. The control group received an equal volume of phosphate buffered saline (PBS). A schematic diagram of the experimental method is shown in Figure 59. Specifically, mice were intraperitoneally injected with each antibody, either alone or in combination, twice a week for a total of four injections over two weeks (Days 12, 15, 19, and 22) (CL7: 10 mg / kg; anti-PD-1 antibody: 10 mg / kg). Tumor volume was measured for 25 days. The results are shown in Figure 60.

[0251] As can be seen from Figure 60, cancer growth was suppressed in the experimental group administered with anti-CD300c monoclonal antibody alone compared to the control group. However, it was confirmed that cancer growth was more effectively suppressed when treated in combination with an immunological anticancer agent such as anti-PD-1 antibody than when treated with anti-CD300c monoclonal antibody alone.

[0252] Experimental Example 9.2. Confirmation of Increased Tumor-Infiltrating Lymphocytes in the In Vivo Tumor Microenvironment To confirm the effect of anti-CD300c monoclonal antibody on tumor-infiltrating lymphocytes (TILs) in the tumor microenvironment (TME), mice were euthanized on day 25 of the experiment using the same method as in Experiment 9.1. After intravenous perfusion with 1% PFA (para-formaldehyde), tumor tissue was obtained. The tumor tissue was fixed using 1% PFA and dehydrated using 10%, 20%, and 30% sucrose solutions. The dehydrated tumor tissue was frozen in an optimal cutting temperature compound (OCT) and then sliced ​​into 50 μm-thick sections using a cryotome. The tissue was incubated in a mixture of 20 mg / ml collagenase D and 2 mg / ml DNase I at 37°C for 1 hour, then filtered through a 70 μm cell strainer to lyse red blood cells and re-filtered through a nylon mesh to disaggregate the cells. To suppress nonspecific reactions in single-cell suspensions, the cells were reacted with CD16 / 32 antibody (obtained from Invitrogen) for 1 hour to confirm cell viability, and tumor-infiltrating lymphocyte markers, CD8+ T cells and CD31+ cancer vascular cells, were stained.

[0253] As a result, it was confirmed that the number of CD8+ T cells increased in the experimental group in which anti-CD300c monoclonal antibody was administered in combination with anti-PD-1 antibody or anti-PD-L1 antibody, anti-CTLA-4 antibody, or anti-CD47 antibody, compared to the experimental group in which anti-CD300c monoclonal antibody was administered alone. This confirmed that when anti-CD300c was administered in combination with immune anticancer agents such as anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, or anti-CD47 antibody, it increased tumor-infiltrating lymphocytes in the tumor microenvironment, resulting in an anti-cancer effect, compared to when anti-CD300c was administered alone.

[0254] Experimental Example 9.3. Confirmation of the effect of increasing M1 macrophages in vivo To confirm whether anti-CD300c monoclonal antibodies increase M1 macrophages in cancer tissue in a mouse model, cancer tissue sections prepared in the same manner as in Experimental Example 9.2 were stained with antibodies against the M1 macrophage marker iNOS and the M2 macrophage marker CD206 and examined by FACS.

[0255] As a result, as shown in Figure 61, the experimental group treated with anti-PD-1 antibody showed a partial increase in M1 macrophages compared to the control group, while the experimental group treated with anti-CD300c monoclonal antibody showed a significant increase in M1 macrophages, with almost no M2 macrophages observed. Furthermore, the experimental group treated with anti-CD300c monoclonal antibody and anti-PD-1 antibody showed a greater increase in M1 macrophages. These results demonstrate that treatment with anti-CD300c monoclonal antibody in combination with immunosuppressive anti-cancer agents such as anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, and anti-CD47 antibody more effectively promotes differentiation into M1 macrophages than treatment with anti-CD300c monoclonal antibody alone.

[0256] Experimental Example 9.4. Confirmation of the in vivo CD8+ T cell immunostimulatory effect To confirm whether the anti-CD300c monoclonal antibody CL7 promotes CD8+ T cell immunity in a mouse tumor model, mice were euthanized on day 25 of the experiment using the same method as in Experimental Example 9.1. After perfusion, 1% PFA was intravenously injected into the mice to obtain tumor tissue. The tumor tissue was fixed using 1% PFA and dehydrated sequentially in 10%, 20%, and 30% sucrose solutions. The dehydrated tumor tissue was frozen in OCT compound and then sliced ​​into 50 μm-thick sections using a frozen tissue sectioner. The tumor tissue was then stained for CD8+ and iNOS.

[0257] As shown in Figure 62, the number of CD8+ T cells in the experimental group treated with anti-PD-1 antibody was slightly increased compared to the control group, but the number of CD8+ T cells in the experimental group treated with anti-CD300c monoclonal antibody was significantly increased. Furthermore, the experimental group treated with anti-CD300c monoclonal antibody and anti-PD-1 antibody showed a greater increase in CD8+ T cells than the group treated with anti-PD-1 alone. These results confirm that the number of CD8+ T cells can be more effectively increased when anti-CD300c monoclonal antibody is used in combination with existing immunotherapy anti-cancer drugs.

[0258] Experimental Example 9.5. Confirmation of the effect of increasing immune cell activity in vivo To determine whether the combination of anti-CD300c monoclonal antibody with an anti-cancer drug increases immune cell activity, spleens were obtained from mice treated with anti-CD300c monoclonal antibody in combination with anti-PD-1, anti-CTLA-4, anti-KIR, anti-LAG3, anti-CD137, anti-OX40, anti-CD276, anti-CD27, anti-GITR, anti-TIM3, anti-41BB, anti-CD226, anti-CD40, anti-CD70, anti-ICOS, anti-CD40L, anti-BTLA, anti-TCR, anti-TIGIT, and anti-CD47 antibodies in the same manner as in Experimental Example 9.1. The obtained spleens were stained by FACS with various markers that indicate T cell activity and NKT cell activity, as described in Experimental Example 9.2, and analyzed by MFI.

[0259] As a result, when anti-CD300c monoclonal antibody was administered in combination with the anticancer immunotherapy, it was confirmed that T cell activation markers Gzma, Icos, CD69, and Ifng increased, and NKT cell activation markers Cd11, CD38, and cxcr6 increased significantly. This confirmed that T cells and NKT cells were more activated when anti-CD300c was administered in combination with the anticancer immunotherapy than when administered alone.

[0260] Experimental Example 9.6. Confirmation of in vivo Treg suppression effect To confirm the changes in Treg cell phenotypes that cause immune suppression when anti-CD300c monoclonal antibody and anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-KIR, anti-LAG3, anti-CD137, anti-OX40, anti-CD276, anti-CD27, anti-GITR, anti-TIM3, anti-41BB, anti-CD226, anti-CD40, anti-CD70, anti-ICOS, anti-CD40L, anti-BTLA, anti-TCR, anti-TIGIT, and anti-CD47 antibodies are administered in combination with solid tumor models, T cells were extracted from spleen tissue prepared in the same manner as in Experiment 9.2, and the number of FOXP3-expressing Treg cells among CD3+ T cells was determined by FACS staining.

[0261] When examining the ratio of Tregs in CD3+ cells, it was confirmed that the ratio of Tregs was significantly reduced when each immunotherapy anti-cancer agent was administered in combination with anti-CD300c monoclonal antibody compared to when each agent was administered alone, indicating that this induces the activation of T cells that attack cancer cells.

[0262] Experimental Example 10: Confirmation of (synergistic) increase in in vivo anti-cancer effect by combined use (melanoma mouse model) Experimental Example 10.1. Confirmation of in vivo tumor growth inhibitory effect To confirm whether the anti-CD300c monoclonal antibody CL7 is effective in other carcinomas besides the CT26 colon cancer mouse model, we conducted additional experiments in a melanoma mouse model. Eight-week-old male C57BL / 6 mice were inoculated with 7x10 B16F10 melanoma cells. 5 Allograft mouse tumor models were constructed by subcutaneous injection of the melanoma cell line. All animal care and experiments were carried out in an SPF facility. Eight days after the melanoma cell line was implanted, tumors grew to 50-100 mm. 3Mice were intraperitoneally injected with 25 mg / kg of CL7, 10 mg / kg of α-PD-1, and 4 mg / kg of α-CTLA-4. A schematic diagram of the experimental method is shown in Figure 63. More specifically, mice were injected twice a week for two weeks for a total of four times with anti-CD300c monoclonal antibody, anti-PD-1 antibody, anti-CD300c monoclonal antibody + anti-PD-1 antibody (combo), and anti-CD300c monoclonal antibody + anti-PD-1 antibody + anti-CTLA-4 antibody (triple). A control group was injected with phosphate buffered saline (PBS) in the same amount as the anti-CD300c monoclonal antibody. Tumor size was measured for 20 days.

[0263] As a result, as shown in Figure 64, it was confirmed that although cancer growth was suppressed by administration of anti-CD300c monoclonal antibody alone, cancer growth was more effectively suppressed in the group in which anti-CD300c monoclonal antibody was administered in combination with anti-PD-1 antibody and anti-CTLA-4 antibody.

[0264] Experimental Example 10.2. Confirmation of increase in cytotoxic T cells To confirm the in vivo effect of the anti-CD300c monoclonal antibody CL7 in combination with anti-PD-1 antibody and / or anti-CTLA-4 antibody on CD8+ T cells in a B16F10 melanoma model, a mouse tumor model was prepared as in Experimental Example 10.1 and each test substance was injected at the same concentration.

[0265] Tumor tissue was collected from six mice per group. After removal, the tumor tissue was incubated in a mixture of collagenase D (20 mg / ml) and DNase I (2 mg / ml) at 37°C for 1 hour. It was then filtered through a 70 μm cell strainer to lyse red blood cells, and then refiltered through a nylon mesh. The single-cell suspension was then blocked with CD16 / 32 antibody (obtained from Invitrogen), and the cells were stained with a cell viability stain and CD8+ and CD4+ antibodies. Data were then read using a CytoFLEX flow cytometer and analyzed using FlowJo software.

[0266] As a result, as shown in Figure 65, similar to the CT26 carcinoma model (Experimental Example 6.2), the number of CD8+ T cells was confirmed to increase in the B16F10 melanoma model by the combined administration of anti-CD300c monoclonal antibody and anti-cancer agent (Groups D and T). Here, Group D represents the combined administration of CL7 and anti-PD-1 antibody, and Group T represents the combined administration of CL7, anti-PD-1 antibody, and anti-CTLA-4 antibody.

[0267] Experimental Example 10.3. Confirmation of an increase in cytotoxic T cells relative to regulatory T cells To confirm the in vivo effect of the anti-CD300c monoclonal antibody CL7 in combination with anti-PD-1 and / or anti-CTLA-4 antibodies on regulatory T cells in a B16F10 melanoma model, a mouse tumor model was prepared as in Experimental Example 10.1 and injected with each test substance at the same concentration. The experimental groups were: (i) a CL7-administered group, (ii) an anti-PD-1-administered group, (iii) a CL7 and anti-PD-1-administered group (Group D), and (iv) a CL7, anti-PD-1, and anti-CTLA-4-administered group (Group T).

[0268] Tumor tissue was collected from six mice per group. After removal, the tumor tissue was incubated in a mixture of collagenase D (20 mg / ml) and DNase I (2 mg / ml) at 37°C for 1 hour. It was then filtered through a 70 μm cell strainer to lyse red blood cells, and then refiltered through a nylon mesh. The single-cell suspension was then blocked with CD16 / 32 (Invitrogen) antibody and stained with a cell viability stain, antibodies against the Treg marker proteins CD25 and Foxp3, CD3+ antibody, and CD8+ antibody. Data were then read using a CytoFLEX flow cytometer and analyzed using FlowJo software.

[0269] As a result, as shown in Figure 66, similar to the CT26 carcinoma model (Experimental Example 6.5), the combined administration of anti-CD300c monoclonal antibody and anti-cancer agent (Groups D and T) also increased CD8+ T cells relative to regulatory T cells in the B16F10 melanoma model. Here, Group D represents the combined use of CL7 and anti-PD-1 antibody, and Group T represents the combined use of CL7, anti-PD-1 antibody, and anti-CTLA-4 antibody.

[0270] Experimental Example 10.4. Confirmation of Increased Tumor-Associated Macrophages (TAM) To confirm the in vivo effect of the combination of anti-CD300c monoclonal antibody CL7 with anti-PD-1 antibody and / or anti-CTLA-4 antibody on macrophages in a B16F10 melanoma model, a mouse tumor model was prepared as in Experimental Example 10.1 and each test substance was injected at the same concentration.

[0271] Tumor tissue was collected from six mice per group. After removal, the tumor tissue was incubated in a mixture of collagenase D (20 mg / ml) and DNase I (2 mg / ml) at 37°C for 1 hour. It was then filtered through a 70 μm cell strainer to lyse red blood cells, and then refiltered through a nylon mesh. The single-cell suspension was then blocked with CD16 / 32 antibody (obtained from Invitrogen), and the cells were stained with a cell viability stain and antibodies against F4 / 80 and iNOS. Data were then read using a CytoFLEX flow cytometer and analyzed using FlowJo software.

[0272] As a result, as shown in Figure 67, similar to the CT26 carcinoma model (Experimental Example 6.1), the combined administration of anti-CD300c monoclonal antibody and anti-cancer agent (Groups D and T) also increased the expression of M1 tumor-associated macrophages in the B16F10 melanoma model. Here, Group D represents the combined administration of CL7 and anti-PD-1 antibody, and Group T represents the combined administration of CL7, anti-PD-1 antibody, and anti-CTLA-4 antibody.

[0273] In summary, the results presented in Figures 65, 66, and 67 have the following implications: Because the anti-CD300c monoclonal antibody CL7 treats cancer in the B16F10 melanoma mouse model using the same mechanism as in the CT26 colon cancer mouse model (Figures 40, 41, and 42), it is predicted that the combined administration of CL7 and an immunosuppressant will have similar effects on various cancers.

[0274] Experimental Example 11: Confirmation of complete remission phenomenon in a mouse model of colon cancer by combined administration To confirm the anti-cancer effect of the combined administration of anti-CD300c monoclonal antibody CL7 and anti-PD-1 antibody and / or anti-CTLA-4 antibody under in vivo conditions, 2x10 colon cancer cell line (CT26) was cultured. 5 The cells were subcutaneously transplanted into 8-week-old BALB / c mice to create an allograft mouse tumor model. All animal care and experiments were carried out in an SPF facility. On day 11 (D11) after colon cancer cell transplantation, tumors grew to 50-100 mm. 3 Mice were administered anti-CD300c monoclonal antibody and anti-PD-1 and anti-CTLA-4 antibodies purchased from BioXcell, either alone or in combination. A control group received phosphate buffered saline (PBS) at the same volume as CL7. Specifically, on days 11, 14, and 18, mice were intraperitoneally injected with each antibody alone or in combination (CL7: 25 mg / kg; anti-PD-1 antibody: 10 mg / kg; anti-CTLA-4 antibody: 4 mg / kg), and tumor volume was measured.

[0275] As a result, as shown in Figure 68a, tumor growth was suppressed in the experimental group administered anti-CD300c monoclonal antibody alone compared to the control group, but tumor growth was more effectively suppressed when administered in combination with immunosuppressive anti-cancer agents such as anti-PD-1 antibody and anti-CTLA-4 antibody than when treated with anti-CD300c monoclonal antibody alone. In particular, tumor size was reduced by 90% in the triple combination administration of CL7 + αPD-1 + αCTLA-4.

[0276] Furthermore, as shown in Figure 68b, when anti-CD300c monoclonal antibody was administered in combination with anti-PD-1 antibody, 50% complete remission (CR) was achieved, and when it was administered in triple combination with anti-CTLA-4 antibody, 70% complete remission (CR) was achieved, confirming that the combination administration resulted in excellent anti-cancer effects.

[0277] Experimental Example 12: Confirmation of the long-term survival rate improvement effect of combined administration The long-term survival rate of mice tested in Experimental Example 11 was examined. As a result, as shown in Figure 69, it was confirmed that the long-term survival rate was improved when treated with anti-CD300c monoclonal antibody in combination with immunosuppressive anti-cancer agents such as anti-PD-1 antibody and anti-CTLA-4 antibody compared to treatment with anti-CD300c monoclonal antibody alone.

[0278] Experimental Example 13: Confirmation of the effect of combined administration on preventing cancer recurrence To confirm the in vivo efficacy of combined administration of anti-CD300c monoclonal antibody CL7 and immunotherapy, 2x10 colon cancer cell line (CT26) was used. 5 The cells were subcutaneously transplanted into 8-week-old BALB / c mice to create an allograft mouse tumor model, and the experiment was carried out as described in Experimental Example 11 to obtain mice that achieved complete remission. The resulting mice were then inoculated with 2 x 10 colon cancer cell line (CT26). 5 The cells were re-transplanted (re-challenge) and observed for 30 days.

[0279] As a result, as shown in Figure 70, it was confirmed that cancer recurrence or metastasis did not occur in the group that achieved complete remission through the combined administration of anti-CD300c monoclonal antibody and anti-cancer immunotherapy. This suggests that individuals who achieved complete remission through the combined administration of anti-CD300c monoclonal antibody and anti-PD-1 antibody (CL7 + αPD-1; Combi) or the triple combination administration in which anti-CTLA-4 antibody was added (CL7 + αPD-1 + αCTLA-4; Triple) will exhibit systemic protective immune responses through sustained immune memory, thereby suppressing cancer recurrence and metastasis.

[0280] Experimental Example 14: Confirmation of immune memory effect by combined administration To analyze effector memory T cells in mice that achieved complete remission in Experiment 13, the mice were sacrificed and their spleens were collected. Splenocytes were then obtained and stained with antibodies against CD44 and CD62L (obtained from Invitrogen), which are markers associated with T cell activity. Data were then read using a CytoFLEX flow cytometer and analyzed using FlowJo software.

[0281] As a result, as shown in Figure 71, it was confirmed that effector memory T cells were significantly increased when anti-CD300c monoclonal antibody and anti-cancer agent were combined. This indicates that, as predicted in Experimental Example 13, mice that achieved complete remission acquired immune memory through the increased effector memory T cells that were obtained when CL7 and anti-PD-1 antibody were combined (Combi) or when an anti-CTLA-4 antibody was added to these antibodies (Triple) and thus suppressed the growth of additional cancer cells.

[0282] Example 4. Combined administration of anti-CD300c monoclonal antibodies (CL10, SL18) and immunosuppressants The anti-CD300c monoclonal antibodies (CL10, SL18) prepared in Example 1 were used in combination with other immunosuppressive anti-cancer drugs, such as the anti-PD-L1 antibody Imfinzi and the anti-PD-1 antibody Keytruda, and the results were observed.

[0283] The sources of these immunotherapy anti-cancer drugs are as follows: Imfinzi (AstraZeneca) and Keytruda (Merck Sharp & Dohme).

[0284] Experimental Example 15. Confirmation of increased M1 macrophage differentiation potential To confirm whether anti-CD300c monoclonal antibodies CL10 or SL18 can promote the differentiation of macrophages into M1 macrophages, 1x10 cells were cultured in a 96-well plate. 4After dispensing THP-1 cells at 1000x / well, they were treated with 10 μg / mL of CL10 or SL18. To confirm the effect of combined treatment with CL10 or SL18 and anticancer drugs, the anti-CD300c monoclonal antibody was also treated with 10 μg / mL of Imfinzi and / or Keytruda. After 48 hours of incubation in a CO2 incubator, the production of TNF-α, a differentiation marker for M1 macrophages, was measured using an ELISA kit (Human TNF-α Quantikine kit, R&D Systems). The results are shown in Figure 72a (combined with CL10) and Figure 72b (combined with SL18).

[0285] As shown in Figures 72a and 72b, TNF-α expression was increased when THP-1 cells were treated with CL10 or SL18 in combination with Imfinzi or Keytruda compared to when treated alone. In particular, the highest TNF-α expression was observed when CL10 or SL18 was administered in combination with the two antibodies, Imfinzi and Keytruda.

[0286] Experimental Example 16: Confirmation of the effect of inhibiting cancer cell growth To confirm the cancer cell growth inhibitory effect of the combined administration of anti-CD300c monoclonal antibodies CL10 or SL18 and immunotherapy, we compared the cell growth inhibitory effect using A549 (human lung cancer cell line) cells. Specifically, in a 96-well plate without FBS, 2 x 10 4 Cells were aliquoted and plated at 6x10 cells per well under 0.1% FBS. 3 The cells were then aliquoted and treated with CL10 or SL18 alone or in combination with Imfinzi and / or Keytruda at a concentration of 10 μg / mL, respectively, and cultured for 5 days. Then, 30 μl of CCK-8 (DOJINDO) was added per well, and the cells were incubated in a CO2 incubator for 4 hours, with absorbance measured at OD 450 nm every hour. The results are shown in Figure 73a (combined with CL10) and Figure 73b (combined with SL18).

[0287] As shown in Figures 73a and 73b, when A549 cells were treated with 0.1% FBS, the cancer cell proliferation inhibitory effect was further enhanced when CL10 or SL18 was treated in combination with Imfinzi or Keytruda compared to when treated alone, and it was confirmed that the cancer cell proliferation inhibitory effect was greatest when CL10 or SL18 was administered in combination with the two antibodies, Imfinzi and Keytruda, simultaneously.

[0288] As can be seen from Experimental Example 15 and this Experimental Example, the remaining anti-CD300c monoclonal antibodies prepared in Example 1, including CL10 and SL18, also exhibited efficacy through the same mechanism of action as CL7, and like CL7, their efficacy was increased when administered in combination with an immunosuppressant.

[0289] statistical processing The results obtained through the experiment were analyzed for comparison between experimental groups using one-way analysis of variance followed by Bonferroni post-hoc test, and the difference between groups was significant when the p-value was 0.05 or less.

Claims

1. (i) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 7, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 8, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 9; The light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 11, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

12. (ii) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 19, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 20, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 21; The light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:22, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:23, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

24. (iii) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 43, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 44, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 45; The light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:46, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:47, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

48. (iv) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 55, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 56, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 57; The light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:58, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:59, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

60. (v) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 67, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 68, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 69; The light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:70, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:71, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

72. (vi) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 79, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 80, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 81; The light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:82, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:83, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

84. (vii) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 103, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 104, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 105; The light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 106, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 107, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

108. (viii) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 115, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 116, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 117; The light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 118, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 119, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

120. (ix) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 127, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 128, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 129; The light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 130, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 131, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

132. (x) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 139, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 140, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 141; The light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 142, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 143, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

144. (xi) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 151, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 152, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 153; The light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 154, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 155, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

156. (xii) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 163, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 164, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 165; The light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 166, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 167, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

168. (xiii) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 199, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 200, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 201; the light chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:202, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:203, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:204; or (xiv) the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 211, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 212, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 213; an anti-CD300c monoclonal antibody or an antigen-binding fragment thereof, wherein the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 214, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 215, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

216.

2. the heavy chain variable region comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 43, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 44, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 45; The anti-CD300c monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 46, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 47, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

48.

3. the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 79, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 80, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 81; The anti-CD300c monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 82, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 83, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

84.

4. the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 115, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 116, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 117; The anti-CD300c monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 118, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 119, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

120.

5. the heavy chain variable region comprises a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 211, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 212, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 213; The anti-CD300c monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region comprises CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 214, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 215, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

216.

6. the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 303, 307, 315, 319, 323, 327, 335, 339, 343, 347, 351, 355, 367, and 371; The anti-CD300c monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 304, 308, 316, 320, 324, 328, 336, 340, 344, 348, 352, 356, 368, and 372.

7. the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 315, 327, 339, and 371; The anti-CD300c monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 316, 328, 340, and 372.

8. The anti-CD300c monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein the anti-CD300c monoclonal antibody or antigen-binding fragment thereof has interspecies cross-reactivity.

9. The anti-CD300c monoclonal antibody or antigen-binding fragment thereof according to claim 8, wherein the anti-CD300c monoclonal antibody or antigen-binding fragment thereof is cross-reactive with both human and mouse CD300c antigens.

10. A pharmaceutical composition for preventing or treating cancer, comprising the anti-CD300c monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 9 as an active ingredient.

11. 11. The pharmaceutical composition of claim 10, wherein the cancer comprises at least one selected from the group consisting of colorectal cancer, colon cancer, thyroid cancer, oral cancer, pharyngeal cancer, laryngeal cancer, cervical cancer, brain cancer, lung cancer, ovarian cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, tongue cancer, breast cancer, uterine cancer, stomach cancer, bone cancer, and blood cancer.

12. The pharmaceutical composition of claim 11, wherein the cancer is a solid cancer.

13. The pharmaceutical composition of claim 11, wherein the cancer comprises one or more selected from the group consisting of colon cancer, lung cancer, melanoma, and breast cancer.

14. The pharmaceutical composition according to claim 11, which suppresses cancer growth, survival, metastasis, recurrence, or resistance to anticancer drugs.

15. The pharmaceutical composition of claim 11 , further comprising one or more immunosuppressive anti-cancer agents.

16. 16. The pharmaceutical composition of claim 15, wherein the immunoanticancer agent comprises at least one selected from the group consisting of anti-PD-1, anti-PD-L1, anti-CTLA-4, anti-KIR, anti-LAG3, anti-CD137, anti-OX40, anti-CD276, anti-CD27, anti-GITR, anti-TIM3, anti-41BB, anti-CD226, anti-CD40, anti-CD70, anti-ICOS, anti-CD40L, anti-BTLA, anti-TCR, and anti-TIGIT.

17. The pharmaceutical composition of claim 15, wherein the immunological anti-cancer agent comprises at least one selected from the group consisting of anti-PD-1, anti-PD-L1, anti-CTLA-4, and anti-CD47 antibodies.

18. The pharmaceutical composition of claim 15, wherein the immunological anti-cancer agent comprises one or more selected from the group consisting of durvalumab, pembrolizumab, nivolumab, αCD47, and ipilimumab.

19. The pharmaceutical composition according to claim 15, wherein the anti-CD300c antibody or its antigen-binding fragment and the anti-cancer immunotherapy agent are each formulated and administered simultaneously or sequentially.

20. A method for preventing or treating cancer, comprising administering the anti-CD300c monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9 to a non-human subject in need of cancer prevention or treatment.

21. 21. The method of claim 20, further comprising administering one or more immune anti-cancer agents.

22. The method of claim 20, further comprising the step of confirming the expression level of CD300c protein based on a biological sample from the subject before administering the anti-CD300c monoclonal antibody or its antigen-binding fragment.

23. The method of claim 22, further comprising a step of confirming the therapeutic reactivity of the anti-CD300c antibody or its antigen-binding fragment based on the expression level of the confirmed marker.

24. The method of claim 20, further comprising determining the expression level of one or more markers selected from the following markers using a biological sample from a subject to which the anti-CD300c monoclonal antibody or its antigen-binding fragment has been administered: Bst2, Cd40, Cd70, Cd86, Ccl8, Xcl1, Ccr7, Cd80, Cd206, Msr1, Arg1, Vegfa, Pdgfrb, Co l4a1, Hif1a, Vcam1, Icam1, Gzma, Gzmb, Icos, Cd69, Ifng, Tnf, Cd1d1, Cd1d2, Cd38, Cxc r6, Xcr1, Tbx21, Stat1, Stat4, Cxcr3, IL-12b, IL-4, IL-6, IL-13, PD-1, PD-L1, CTLA- 4, Lag3, Tim3, Icos, Ox40, Gitr, Hvem, CD27, CD28, Cma1, Timd4, Bcl6, Cxcl5 and Ccl21a.

25. The method of claim 24, further comprising selecting one or more immunological anti-cancer agents to be used in combination with the anti-CD300c monoclonal antibody or its antigen-binding fragment based on the expression level of the identified marker.

26. 26. The method of claim 25, wherein the marker comprises one or more selected from the group consisting of PD-1, PD-L1, CTLA-4, Lag3, Tim3, Icos, Ox40, Gitr, Hvem, CD27, and CD28.

27. 25. The method of claim 24, wherein the marker comprises one or more selected from the group consisting of vegfa, pdgfrb, Col4a1, Hif1a, Bst2, CCL8, Xcl1, CCR7, CD80, Tbx21, Stat1, Stat4, Ifng, Cxcr3, IL-6, Gzma, Icos, Cd69, Cd1d1, Cd38, Cxcr6, Ox40, Gitr, CD27, and CD28.

28. The method of claim 27, further comprising determining that the therapeutic responsiveness to the anti-CD300c antibody or its antigen-binding fragment is good or excellent when the expression level of one or more markers selected from the group consisting of vegfa, pdgfrb, Col4a1, Hifla, and IL-6 is statistically significantly reduced compared to a subject not administered the anti-CD300c antibody or its antigen-binding fragment.

29. The method of claim 27, further comprising determining that the therapeutic responsiveness of the anti-CD300c antibody or its antigen-binding fragment is good or excellent when the expression level of one or more markers selected from the group consisting of Bst2, CCL8, Xcl1, CCR7, CD80, Tbx21, Stat1, Stat4, Ifng, Cxcr3, Gzma, Icos, Cd69, Cd1d1, Cd38, Cxcr6, Ox40, Gitr, CD27, and CD28 is statistically significantly increased compared to a subject not administered the anti-CD300c antibody or its antigen-binding fragment.

30. A composition comprising the anti-CD300c monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9, and and instructions for use of said antibody or antigen-binding fragment thereof. Cancer prevention or treatment kits.

31. 31. The kit of claim 30, wherein the instructions include instructions for use of a combination of the antibody or antigen-binding fragment thereof and one or more additional anti-cancer agents.

32. The kit of claim 30, wherein the instructions include instructions for measuring the expression level of CD300c protein using a biological sample obtained from the subject prior to administration of the antibody or its antigen-binding fragment.

33. measuring the expression level of CD300c protein based on a biological sample obtained from a subject in need of cancer prevention or treatment; The information for preventing or treating cancer includes information on the therapeutic reactivity of the anti-CD300c monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 9, or information on the selection of the anti-CD300c monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 9. A method for providing information for the prevention or treatment of cancer.

34. The present invention includes a substance for measuring the expression level of CD300c protein using a biological sample obtained from a subject in need of cancer prevention or treatment, A composition comprising the anti-CD300c monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9. A kit for providing information for the prevention or treatment of cancer.

35. An isolated nucleic acid molecule encoding the anti-CD300c monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

36. 36. An expression vector comprising the nucleic acid molecule of claim 35.

37. A host cell comprising the nucleic acid molecule of claim 35.

38. A method for producing an anti-CD300c monoclonal antibody or an antigen-binding fragment thereof, comprising culturing the host cell of claim 35.

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