JP2024517985A - Anti-CD300c monoclonal antibody and its biomarker for preventing or treating cancer - Google Patents

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 against 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

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, which contain the same.

Cancer is one of the diseases that accounts for the largest proportion of deaths in modern people. It is a disease that occurs when normal cells change due to gene mutations caused by various factors, and refers to a malignant tumor that does not follow the differentiation, proliferation, and growth morphology of normal cells. Cancer is characterized by "uncontrolled cell growth", and such abnormal cell growth forms a cell mass called a tumor, which penetrates into surrounding tissues and, in severe cases, may metastasize to other organs in the body. Cancer is an intractable chronic disease that in many cases cannot be fundamentally cured even when treated with surgery, radiation, and drug therapy, causing pain to patients and ultimately leading to death. In particular, the incidence of cancer in the world has been increasing by more than 5% every year due to the increase in the elderly population and environmental deterioration, and according to a WHO report, it is estimated that the number of people with cancer will increase to 30 million within the next 25 years, of which 20 million will die from cancer.

Cancer drug therapy, i.e., anticancer drugs, generally treat cancer by attacking and killing cancer cells as cytotoxic compounds, but they cause severe 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 such targeted anticancer drugs have reduced side effects, they have a high probability of causing resistance, which is a limiting factor. Therefore, in recent years, interest in immune anticancer drugs that use the body's immune system to reduce problems caused by toxicity and resistance has been increasing rapidly. As an example of such immune anticancer drugs, immune barrier inhibitors have been developed that specifically bind to PD-L1 on the surface of cancer cells, inhibit the binding of T cells to PD-1, and activate T cells to attack cancer cells. However, such immune barrier inhibitors are not effective for a wide variety of cancers, so there is a pressing need to develop new anti-inflammatory immune therapeutic agents that are equally effective for a wide variety of cancers.

Korean Patent Publication No. 10-2018-0099557

The purpose of this invention is to solve all of the above problems.

One object of the present invention is to provide an anti-CD300c antibody for the prevention or treatment of cancer.

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

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.

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

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.

The object of the present invention is not limited to the object mentioned above. The object of the present invention will become clearer in the following description and is realized by the means and combinations thereof described in the claims.

The representative configuration of the present invention to achieve the above objective is as follows:

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

According to another aspect of the present invention, there is provided an anti-CD300c antibody or an antigen-binding fragment thereof and its use for preventing or treating cancer.

According to another aspect of the present invention, there is provided a use of an anti-CD300c antibody or an antigen-binding fragment thereof for the manufacture of a drug for the prevention or treatment of cancer.

According to another aspect of the present invention, there is provided an anti-cancer therapy comprising an anti-CD300c antibody or an antigen-binding fragment thereof as an active ingredient.

According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer, comprising an anti-CD300c antibody or an antigen-binding fragment thereof as an active ingredient.

According to another aspect of the present invention, there is provided a method for preventing or treating cancer, comprising administering an anti-CD300c antibody or an antigen-binding fragment thereof to a subject in need of such prevention or treatment.

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 antigen-binding fragment thereof, and instructions for using the antibody or antigen-binding fragment thereof.

The anti-CD300c monoclonal antibody according to the present invention binds specifically and with high binding strength to CD300c expressed on the surface of various cancers, activating T cells and promoting differentiation into M1 macrophages, thereby effectively suppressing the proliferation of cancer cells and can be effectively used as an immunotherapeutic agent for various cancers. In addition, the anti-CD300c monoclonal antibody according to the present invention not only further increases its therapeutic effect when administered in combination with existing immunological anticancer drugs, but also has cross-reactivity between species, making it widely applicable to various mammals. In addition, when the anti-CD300c monoclonal antibody according to the present invention is administered to resistant cancer cells that exhibit the ability to resist apoptosis, it is expected to significantly weaken the resistance of the cancer cells and show excellent efficacy in preventing cancer recurrence. In addition, cancer cells generally evade the immune system by inhibiting the production of IL-2, a proinflammatory cytokine. Anti-CD300c monoclonal antibodies have been shown to restore the production of IL-2 blocked by such cancer cells, thereby inducing the death of cancer cells through an activated immune system, and are expected to be used as a more fundamental immune anticancer agent.

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 sites (CDR1, CDR2, and CDR3) are indicated in order. FIG. 1 is a schematic diagram showing the mechanism by which the anti-CD300c monoclonal antibody and/or CD300c siRNA of the present invention exerts an anti-cancer effect. FIG. 1 is a schematic diagram showing the mechanism by which the anti-CD300c monoclonal antibody of the present invention acts on monocytes, T cells, and cancer cells, respectively. 1 shows the results of SDS-PAGE under non-reducing conditions for the anti-CD300c monoclonal antibody according to Example 1.4. 1 shows the results of SDS-PAGE under reducing conditions of the anti-CD300c monoclonal antibody according to Example 1.4. 1 shows the results of comparing the expression of CD300c in normal cells, immune cells, and cancer cell lines according to Experimental Example 1.1. 1 shows the results of confirming the expression of CD300c in cancer tissues according to Experimental Example 1.2. 1 shows the results of confirming the expression of CD300c in immune cells according to Experimental Example 1.2. 1 shows the results of confirming the expression of CD300c in tonsillar tissue in Experimental Example 1.3. 1 shows the results of confirming the expression of CD300c in cancer tissues according to Experimental Example 1.3. 1 shows the results of confirming the binding ability of anti-CD300c monoclonal antibody to the CD300c antigen in Experimental Example 2.1. 1 shows a sigmoidal curve resulting from FACS binding in Experimental Example 2.2. 1 shows the results of binding ELISA according to Experimental Example 2.3. 1 shows the results of surface plasmon resonance according to Experimental Example 2.4. 1 shows the results of binding ELISA according to Experimental Example 2.5. 1 shows the results of binding ELISA according to Experimental Example 2.6. In relation to Experimental Example 2.7, the results of comparing overall survival in patients with various cancers according to CD300c expression levels are shown. 3 shows the results of confirming the anti-cancer effect of anti-CD300c monoclonal antibody through T cell activation in Experimental Example 3.1. The results of Experimental Example 3.2 confirming the effect of anti-CD300c monoclonal antibody on differentiation into M1 macrophages are shown. 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. The results of Experimental Example 3.4 confirming the effect of anti-CD300c monoclonal antibody on differentiation into M1 macrophages are shown. The results of reconfirming whether anti-CD300c monoclonal antibody promotes differentiation of human monocytes into M1 macrophages in Experimental Example 3.5 are shown below. 13 shows the results of confirming whether anti-CD300c monoclonal antibody in Experimental Example 3.6 redifferentiates M2 macrophages into M1 macrophages. 1 shows the results of confirming the differentiation and re-differentiation ability of anti-CD300c monoclonal antibody into M1 macrophages in Experimental Example 3.7. 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. 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. The results of Experimental Example 4.2 are shown to confirm the effect of inhibiting the growth of cancer cells depending on the concentration of anti-CD300c monoclonal antibody. The results of Experimental Example 4.3 confirming whether anti-CD300c monoclonal antibody promotes differentiation of mouse macrophages into M1 macrophages are shown. The results of Experimental Example 4.4 confirming whether the anti-CD300c monoclonal antibody has an anti-cancer effect are shown. The results of comparing the M1 macrophage differentiation ability of anti-CD300c monoclonal antibody and existing immunological anticancer agents according to Experimental Example 5.1 are shown. The results of comparing the M1 macrophage differentiation ability of anti-CD300c monoclonal antibody and existing immunological anticancer agents according to Experimental Example 5.1 are shown. The results of comparing the M1 macrophage differentiation ability of anti-CD300c monoclonal antibody and existing immunological anticancer agents according to Experimental Example 5.1 are shown. The results of comparing the M1 macrophage differentiation ability of anti-CD300c monoclonal antibody and existing immunological anticancer agents according to Experimental Example 5.1 are shown. The results of comparing the differentiation ability of M0 macrophages to M1 macrophages between anti-CD300c monoclonal antibody and existing immunological anticancer agents according to Experimental Example 5.2 are shown. The results of Experimental Example 5.3 show a comparison of the differentiation ability into M1 macrophages between anti-CD300c monoclonal antibody and existing immunological anticancer agents. The results of comparing the cancer cell growth inhibitory effects between anti-CD300c monoclonal antibody and existing immunological anticancer drugs according to Experimental Example 5.4 are shown below. The results of comparing the cancer cell growth inhibitory effects between anti-CD300c monoclonal antibody and existing immunological anticancer drugs according to Experimental Example 5.4 are shown below. The results of Experimental Example 6.1 showing the effect of anti-CD300c monoclonal antibody on tumor-associated macrophages under in vivo conditions are shown. The results of Experimental Example 6.2 showing the effect of anti-CD300c monoclonal antibody on CD8+ T cells under in vivo conditions are shown. The results of Experimental Example 6.3 are shown to confirm whether anti-CD300c monoclonal antibody increases the number of CD8+ T cells in a tumor-specific manner. 6 shows the results of confirming the effect of anti-CD300c monoclonal antibody on increasing the activity of CD8+ T cells under in vivo conditions according to Experimental Example 6.4. 6 shows the results of in vivo confirmation of the effect of anti-CD300c monoclonal antibody on increasing cytotoxic T cells compared to regulatory T cells according to Experimental Example 6.5. Experimental Example 6.6 shows the results of confirming the effects of anti-CD300c monoclonal antibody on cytotoxic T cells, regulatory T cells, and tumor-associated macrophages. The results of Experimental Example 6.7 confirming the anti-cancer effect of anti-CD300c monoclonal antibody under in vivo conditions are shown below. FIG. 2 shows the results of Nanostring Immune profiling obtained when a solid tumor model was treated with CL7 according to Example 2.1. 2 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. The changes in expression of immune barrier markers confirmed based on the Nanostring immune profiling results obtained in Example 2.1 according to Example 2.2 are shown. * indicates markers whose expression levels were statistically significantly changed compared to before CL7 treatment. 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 drug, either alone or in combination, according to Experimental Example 7.1. 1 shows the results of differentiation of monocytes into M1 macrophages by treatment with anti-CD300c monoclonal antibody in Experimental Example 7.2 (showing the presence or absence of an increase in M1 macrophage markers). 1 shows the results of differentiation of monocytes into M1 macrophages by combined treatment of anti-CD300c monoclonal antibody and an immunological anticancer agent in Experimental Example 7.2 (showing the presence or absence of an increase in M1 macrophage markers). The results of Experimental Example 7.4 show that signal transduction of MAPK, which is a signal for M1 macrophage differentiation, was confirmed when anti-CD300c monoclonal antibody and an immunological anticancer drug were combined. The results of Experimental Example 7.4 confirming the signal transduction of NF-kB, which is a signal for M1 macrophage differentiation, when anti-CD300c monoclonal antibody and an immunological anticancer drug were combined. The results of Experimental Example 7.4 confirming the signal transduction of IkB, which is a signal for M1 macrophage differentiation, when anti-CD300c monoclonal antibody and an immunological anticancer drug were treated in combination. The results of Experimental Example 8.1 show changes in cell apoptosis signals observed when anti-CD300c monoclonal antibody and an immunosuppressant were administered in combination. The results of Experimental Example 8.2 show the effect of inhibiting the growth of cancer cells when anti-CD300c monoclonal antibody and an immunological anticancer drug are combined. The results of Experimental Example 8.2 show the effect of inhibiting the growth of cancer cells when anti-CD300c monoclonal antibody and an immunological anticancer drug are combined. 9 shows a schematic diagram of the experimental method used in Example 9.1. The in vivo cancer 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 is shown. The results of Experimental Example 9.3 are shown to confirm whether anti-CD300c monoclonal antibody increases M1 macrophages in cancer tissues in a mouse model. The results of Experimental Example 9.4 confirming whether anti-CD300c monoclonal antibody promotes CD8+ T cell immunity in a mouse tumor model are shown. 10 shows a schematic diagram of the experimental method used in Example 10.1. The results of confirming whether the anti-CD300c monoclonal antibody according to Experimental Example 10.1 is effective on other carcinomas besides the CT26 colon cancer mouse model are shown below. The results of in vivo examination of the effect of single or combined administration (including double and triple combined administration) of anti-CD300c monoclonal antibody and immuno-anticancer agent in Experimental Example 10.2 on CD8+ T cells in B16F10 melanoma model are shown. The results of Experimental Example 10.3 show the effect of single or combined administration (including double and triple combined administration) of anti-CD300c monoclonal antibody and immuno-anticancer agent on regulatory T cells in a B16F10 melanoma model under in vivo conditions. The results of in vivo examination of the effect of single or combined administration (including double and triple combined administration) of anti-CD300c monoclonal antibody and immuno-anticancer agent in Experimental Example 10.4 on macrophages in a B16F10 melanoma model are shown. 1 shows the results of confirming the anti-cancer effect under in vivo conditions by combined administration of anti-CD300c monoclonal antibody and an immunological anti-cancer agent according to Experimental Example 11. The tumor volume reduction rate is shown. 1 shows the results of confirming the anti-cancer effect of combined administration of anti-CD300c monoclonal antibody and an immunological anti-cancer agent under in vivo conditions according to Experimental Example 11. The complete remission rate is shown. 13 shows the results of Experimental Example 12 confirming the effect of improving long-term survival rate by combined administration of anti-CD300c monoclonal antibody and an immunological anticancer agent. 13 shows the results of confirming the effect of preventing cancer recurrence by combined administration of anti-CD300c monoclonal antibody and an immunological anticancer agent under in vivo conditions. 1 shows the results of confirming the immune memory effect caused by combined administration of anti-CD300c monoclonal antibody and an immunological anticancer agent according to Experimental Example 14. 15 shows the results of confirming whether single or combined administration (including double and triple combined administration) of anti-CD300c monoclonal antibody and an immunological anticancer agent promotes differentiation of monocytes into M1 macrophages. The results of Experimental Example 16 confirming whether the anti-CD300c monoclonal antibody can suppress the growth of cancer cells when administered in combination with an immunosuppressant. The results of Experimental Example 16 confirming whether the anti-CD300c monoclonal antibody can suppress the growth of cancer cells when administered in combination with an immunosuppressant.

The detailed description of the present invention described below will be described with reference to certain drawings of specific embodiments in which the present invention may be practiced, but the present invention is not limited thereto, but only by the appended claims together with the full scope of equivalents thereto, if properly described. It should be understood that the various embodiments of the present invention, although different, are not necessarily mutually exclusive. For example, the specific shapes, structures, and characteristics described herein may be modified from one embodiment to another or embodied in combination without departing from the spirit and scope of the present invention. The technical and scientific terms used herein shall have the same meaning as commonly used in the field 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.

Definitions The term "about" as used herein refers to the normal range of error for the respective value known to one of ordinary skill in the art.

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 or antibody-compound fusions), and antibody fragments (including antigen-binding fragments). As used herein, the prefix "anti-" when referring to an antigen means 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, but not limited to, screening of recombinant antibody libraries with phage or similar vectors, or by immunization of animals with the antigen or antigen-encoding nucleic acid. 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 the 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 referred to as CDR1, CDR2 and CDR3. The more conserved regions in the variable regions outside the CDRs are referred to as "framework regions" ("FRs"). In this specification, the antibody may be, for example, an animal antibody, a chimeric antibody, a humanized antibody or a human antibody.

The term "humanization" (also called reshaping or CDR grafting) includes established techniques to reduce the immunogenicity and improve the affinity or effector functions (ADCC, complement activation, Clq binding) of monoclonal antibodies from xenogeneic sources (usually rodent).

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 of the population are identical except for natural mutations and/or post-translational modifications (e.g., isomerization, amidation) which may be present in minor amounts. Monoclonal antibodies are highly specific and directed against one antigenic site. Monoclonal antibodies exhibit the characteristics of antibodies obtained from a substantially homogeneous population and are 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 techniques, and methods utilizing transgenic animals containing all or a portion of the human immunoglobulin locus.

The term "antigen-binding fragment" refers to a portion of an antibody that has specific binding ability to an antigen, or a polypeptide comprising the same. "Antibody" and "antigen-binding fragment" are used interchangeably, except where "antibody" is specifically understood to exclude "antigen-binding fragment" in the context, and "antibody" may be interpreted to include "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.

The term "anticancer agent" refers collectively to known drugs used in existing cancer treatments that exert cytotoxic or cytostatic effects on cancer cells by acting on various metabolic pathways in cells, and includes chemical anticancer agents, targeted anticancer agents, and immunological anticancer agents.

The term "immunotherapy" refers to drugs that activate immune cells to kill cancer cells.

The term "subject" is used interchangeably with "patient" and may refer to a mammal in need of cancer prevention or treatment, such as primates (e.g., humans), pet animals (e.g., dogs, cats, etc.), livestock animals (e.g., cows, pigs, horses, sheep, goats, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.). In one embodiment of the present invention, the subject is a human.

The term "treatment" generally means 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, prevention of disease onset or recurrence, amelioration of symptoms, reduction of any direct or indirect pathological consequences of a disease, prevention of metastasis, reduction in the rate of disease progression, amelioration or alleviation of the disease state, and remission or improved prognosis. Preferably, "treatment" may refer to medical intervention of an already manifested disease or disorder.

The term "prevention" relates to prophylactic treatment, i.e. measures or procedures aimed at preventing rather than treating 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.

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 objective (e.g., prevention or treatment of cancer).

The term "biological sample" encompasses a variety of sample types obtained from a subject and may be used in a diagnostic or monitoring assay. 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 said biological sample.

The term "expression level" can be determined by measuring one or more expression levels in the mRNA and protein of the marker, and any method known in the art can be used to measure the expression level of the mRNA or protein. For example, the preparation for measuring the expression level of the mRNA can be a primer pair or a probe that specifically binds to the marker gene, and the preparation for measuring the expression level of the protein can be an antibody, substrate, ligand, or cofactor that specifically binds to the marker. Analytical methods for measuring the expression level of the mRNA 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 the protein level 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.

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

Anti-CD300c Antibody According to one aspect of the present invention, there is provided an anti-CD300c antibody or an antigen-binding fragment thereof. The anti-CD300c antibody or an 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 an antigen-binding fragment thereof is a monoclonal antibody or an antigen-binding fragment thereof that specifically binds to CD300c protein.

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

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

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

In one embodiment, 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, as compared to the average CD300c expression level of cancer patients, have a shorter survival time than cancer patients with a low CD300c expression level. This means that inhibition of the expression or activity of CD300c using the anti-CD300c antibody or antigen-binding fragment thereof according to the present invention brings about a cancer treatment effect or an effect of increasing the survival time of cancer patients.

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 can activate T cells and promote differentiation into M1 macrophages, thereby effectively suppressing the proliferation of cancer cells, which allows the anti-CD300c antibody to be effectively used as an immunotherapeutic agent for various cancers. In addition, the therapeutic effect of the anti-CD300c antibody can be further increased by co-administration with existing anti-cancer drugs, and since the anti-CD300c antibody has interspecies cross-reactivity (e.g., human antigen and mouse antigen), it can be widely applied to various mammals. In addition, when the anti-CD300c antibody is administered to resistant cancer cells that exhibit the ability to resist apoptosis, it is expected to show excellent efficacy in preventing the recurrence of cancer by significantly weakening the resistance of the cancer cells. In addition, cancer cells generally evade the immune system by inhibiting the production of IL-2, a proinflammatory cytokine. Anti-CD300c antibodies have been shown to induce cancer cell death through an activated immune system by restoring the amount of IL-2 production blocked by such cancer cells. Therefore, it is expected that they can be used as a more fundamental immune anti-cancer drug. For details regarding CD300c protein or anti-CD300c antibodies, please refer to Korean Patent Publication No. 10-2019-0136949, the contents of which are incorporated herein in their entirety.

In one embodiment, the anti-CD300c monoclonal antibody or antigen-binding fragment thereof 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: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 present invention may comprise a light chain variable region comprising 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.

In further 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 comprises:
(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 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: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.

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 comprises:
(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 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.

In another embodiment, the heavy chain variable region may include a CDR1 comprising or configured as the amino acid sequence shown in SEQ ID NO: 43, a CDR2 comprising or configured as the amino acid sequence shown in SEQ ID NO: 44, and a CDR3 comprising or configured as the amino acid sequence shown in SEQ ID NO: 45, and the light chain variable region may include a CDR1 comprising or configured as the amino acid sequence shown in SEQ ID NO: 46, a CDR2 comprising or configured as the amino acid sequence shown in SEQ ID NO: 47, and a CDR3 comprising or configured as the amino acid sequence shown in SEQ ID NO: 48.

In another embodiment, the heavy chain variable region may include a CDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO:79, a CDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO:80, and a CDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO:81, and the light chain variable region may include a CDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO:82, a CDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO:83, and a CDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO:84.

In another embodiment, the heavy chain variable region may include a CDR1 that includes or is composed of the amino acid sequence shown in SEQ ID NO: 115, a CDR2 that includes or is composed of the amino acid sequence shown in SEQ ID NO: 116, and a CDR3 that includes or is composed of the amino acid sequence shown in SEQ ID NO: 117, and the light chain variable region may include a CDR1 that includes or is composed of the amino acid sequence shown in SEQ ID NO: 118, a CDR2 that includes or is composed of the amino acid sequence shown in SEQ ID NO: 119, and a CDR3 that includes or is composed of the amino acid sequence shown in SEQ ID NO: 120.

In another embodiment, the heavy chain variable region may include a CDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO:211, a CDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO:212, and a CDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO:213, and the light chain variable region may include a CDR1 comprising or consisting of the amino acid sequence shown in SEQ ID NO:214, a CDR2 comprising or consisting of the amino acid sequence shown in SEQ ID NO:215, and a CDR3 comprising or consisting of the amino acid sequence shown in SEQ ID NO:216.

In another embodiment, the heavy chain variable region may comprise 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 may comprise 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.

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

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

FTFX1X2X3X4MX5WVR (1) (SEQ ID NO: 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 not present X9 = R, S, G, or not present X10 = N or not present X11 = Y or not present 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) (SEQ ID NO: 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.

In certain embodiments, the anti-CD300c antibody or antigen-binding fragment may include 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 sequence or the sequences presented in Tables 3, 4, and 5 below.

In certain embodiments, amino acid sequence variants of the antibodies of the invention are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. 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 amino acid sequence of the antibody, and/or insertion of residues into and/or substitution of residues within such amino acid sequences. Any combination of various modifications including deletion, insertion and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired properties, e.g., antigen binding properties. Sites of interest for the induction of substitution mutagenesis include the heavy chain variable region (HVR) and framework regions (FR). Conservative substitutions are provided under the heading of "preferred substitutions" in Table 1 and are further described below in connection with amino acid side chain classes (1)-(6). Amino acid substitutions can be introduced into the molecule of interest and the products screened for a desired activity, e.g., maintained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

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 that affect chain orientation: Gly, Pro;
(6) Aromatic: Trp, Tyr, Phe.

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

As used herein, the term "amino acid sequence variant" includes substantial variants in which there is an amino acid substitution in 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 are those that have/have a particular biological property modification, e.g., an improvement (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody-binding molecule or that substantially maintain a particular biological property of the parent antigen-binding molecule. An exemplary substitution variant is an affinity matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques known in the art. Briefly, one or more HVR residues are mutated, and the variant antigen-binding molecules are displayed on phage and screened for a particular 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 (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made in HVRs.

Amino acid sequence insertions can include intrasequence insertions of single or multiple amino acid residues as well as amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more 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 fusion to the N- or C-terminus of a polypeptide that increases the serum half-life of the antibody. Additionally, other insertional variants of the molecule can include the fusion to the N- or C-terminus of a polypeptide to facilitate crossing the blood-brain barrier (BBB).

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 produced by a variety of techniques, including CDR mutation (Yang et al., J. Mol. Biol., 254, 392-403, 1995), chain shuffling (Marks et al., Bio/Technology, 10, 779-783, 1992), and the use of the E. These have been obtained by a number of affinity maturation protocols, including 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., Nature, 391, 288-291, 1998). , Science, 239, 1534-1536, 1988] discusses such affinity maturation methods.

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.

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

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. In this specification, the antibody-drug conjugate refers to a form in which a drug is bound to an amino acid residue at the N-terminus of the heavy and/or light chain of an antibody, specifically, a form in which a drug is bound to the α-amine group at the N-terminus of the heavy and/or light chain of an antibody.

The term "drug" can refer to any substance that has a specific biological activity against cells (e.g., cancer cells), and includes DNA, RNA, and peptides. The drug may be in a form that includes a reactive group that can react with an α-amine group to form crosslinks, and also includes a form in which a linker that includes a reactive group that can react with an α-amine group to form crosslinks is linked.

The reactive group capable of reacting with the α-amine group to crosslink is not particularly limited in type as long as it can react with the α-amine group at the N-terminus of the heavy or light chain of an antibody to crosslink, and includes all types known in the art that react with amine groups. Examples of the compound include isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxal, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters, but are not limited thereto.

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, but may preferably be an anticancer drug.

Nucleic Acids, Vectors, Host Cells and Methods of Production The anti-CD300c monoclonal antibodies or antigen-binding fragments thereof of the invention can be produced by any antibody production technique known in the art.

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 an antigen-binding fragment thereof. Such a nucleic acid molecule may encode an amino acid sequence including a heavy chain variable region or a heavy chain CDR region and/or an amino acid sequence including a light chain variable region or a light chain CDR region of the anti-CD300c monoclonal antibody. For the sequences of the nucleic acid molecule encoding the heavy/light chain variable regions and CDR regions of the anti-CD300c monoclonal antibody according to the present invention, see Tables 3 to 5 and FIG. 1.

The term "nucleic acid molecule" refers to a nucleic acid molecule that includes DNA (gDNA and cDNA) and RNA molecules, and the nucleotides that are the basic building blocks of nucleic acid molecules include not only natural nucleotides but also analogues in which the sugar or base site is modified. The sequences of the nucleic acid molecules encoding the heavy chain variable region, light chain variable region, and CDR region of the present invention may be modified. The modifications include addition, deletion, or non-conservative or conservative substitution of nucleotides. The nucleic acid molecules of the present invention are also understood to include nucleotide sequences that show substantial identity to the nucleotide sequence. The substantial identity refers to a nucleotide sequence that shows 80% or more homology, in one specific example, 90% or more homology, in another specific example, 95% or more homology, and in another specific example, 98% or more homology, when the nucleotide sequence of the present invention is aligned with any other sequence so as to correspond to the maximum extent possible and the aligned sequences are analyzed using an algorithm commonly used in the art.

According to another aspect of the invention, there is provided one or more vectors (e.g., expression vectors) comprising the nucleic acid.

The term "vector" refers to a nucleic acid molecule capable of carrying 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 integrate into the genome of the host cell upon introduction into the host cell, and thereby be replicated along with the host genome. In addition, certain vectors are capable of inducing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors." Conventional 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 that serve equivalent functions, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses).

According to another aspect of the invention, there is provided a host cell comprising one or more nucleic acid molecules (e.g., polynucleotides) encoding a monoclonal antibody of the invention.

The host cell may be a cell transformed with a recombinant vector of the 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, Serratia marcescens, and various Pseudomonas species. Suitable eukaryotic host cells to be transformed 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" is also used to refer to a transformed cell or a cell capable of expressing a selected gene of interest after transformation with a nucleic acid sequence. The term includes the progeny of a mother cell, whether or not the progeny is identical in morphology or genetic make-up to the original parent, so long as the selected gene is present.

According to another aspect of the present invention, there is provided a method for producing an anti-CD300c monoclonal antibody or an antigen-binding fragment thereof, comprising the step of culturing the host cell.

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

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

The present inventors have confirmed that an anti-CD300c antibody or antigen-binding fragment thereof exerts an enhanced anti-cancer effect when used in combination with one or more other immunological anti-cancer agents. Thus, the anti-CD300c antibody or antigen-binding fragment thereof of the present invention is used in combination with one or more other immunological anti-cancer agents to prevent or treat cancer.

Immuno-anticancer drugs have the advantage of being widely used for many cancers even without specific genetic mutations, since they have a new mechanism for activating the body's immune cells to kill cancer cells. In addition, immuneo-anticancer drugs have fewer side effects as they treat cancer by strengthening the patient's own immune system, and they have the effect of improving the quality of life of patients and significantly extending their survival time. Such immuneo-anticancer drugs include immune barrier inhibitors, and may be manufactured by known methods or may be commercially available products. Examples of immune anti-cancer agents 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 immune anticancer 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).

In one embodiment, the immunological anti-cancer agent may include 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. As an example, the immunological anti-cancer agent may include one or more selected from the group consisting of anti-PD-1, anti-PD-L1, anti-CTLA-4, and anti-CD47 antibodies.

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

According to another aspect of the present invention, there is provided a method for preventing or treating cancer , ameliorating or reducing the severity of at least one symptom or sign of cancer, inhibiting metastasis, or inhibiting the growth of cancer in a subject 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 proliferation, survival, metastasis, recurrence, or resistance to anti-cancer drugs. Such a method may include the step of 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.

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 colorectal cancer, small intestinal cancer, colon cancer, rectal cancer, colon cancer, thyroid cancer, endocrine cancer, oral cancer, tongue cancer, pharyngeal cancer, laryngeal cancer, esophageal cancer, cervical cancer, uterine cancer, fallopian tube cancer, ovarian cancer, brain cancer, head and neck cancer, lung cancer, lymphatic cancer, gallbladder cancer, bladder cancer, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer (or melanoma), breast cancer, stomach cancer, bone cancer, blood cancer, etc., depending on the site of onset, but may include all cancers as long as they express CD300c protein on the surface of the cancer cells. In one embodiment, the cancer may include 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. In another embodiment, the cancer may be a solid cancer.

In one embodiment, the method may further include 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.

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

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

In other embodiments, the method may further include administering one or more immunological anti-cancer agents. When (i) the 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.

"Administered sequentially" means that one component is administered and then another component is administered immediately or at a certain interval thereafter, and the components can be administered in any order. That is, one or more immunological anticancer agents can be administered immediately 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 administration of an anti-CD300c antibody or antigen-binding fragment thereof, followed by the administration of another one of the one or more immunological anticancer agents.

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

Each of the antibody or antigen-binding fragment thereof according to the present invention and, optionally, one or more additional anti-cancer agents can be administered in various ways depending on whether local or systemic treatment is desired and the area to be treated. The method of administering such 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 can be oral, parenteral, inhalation, topical or local administration (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 administration (e.g., intracerebroventricular administration). In addition, when used in combination, the anti-CD300c antibody and the additional immunological anti-cancer agent can be administered by the same route or by different routes.

In the above method, the effective amount of each 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 generally, about 0.01 mg to 100 mg, or 5 mg to about 50 mg per kg of body weight, may be administered once or several times a day. However, the scope of the present invention is not limited to these amounts, as they may increase or decrease depending on the route and period of administration, severity of the disease, sex, weight, age, etc.

The method according to the present invention may include a step of confirming the expression level of CD300c protein in advance from a subject. Whether or not to administer an anti-CD300c antibody or an antigen-binding fragment thereof can be determined based on the expression level.

In another embodiment, the method according to the present invention may include a step of selecting an additional (one or more) immunological anticancer agent suitable for use in combination with the anti-CD300c antibody or antigen-binding fragment thereof by measuring changes in the expression levels of specific markers after administering the anti-CD300c antibody or antigen-binding fragment thereof to a subject.

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

Bst2, Cd40, Cd70, Cd86, Ccl8, Xcl1, Ccr7, Cd80, Cd206, Msr1, Arg1, Vegfa, Pdgfrb, Col4a1, Hif1a, Vcam1, Icam1, Gzma, Gzmb, Icos, Cd69, Ifng, Tnf, Cd1d1, Cd1d2, Cd38, C xcr6, 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.

For an explanation of the markers, see Table 2 below.

In another embodiment, the method may further include a step of selecting an additional immunological anticancer agent based on the expression level of the identified marker. In this case, the marker may include, but is not limited to, PD-1, PD-L1, CTLA-4, Lag3, Tim3, Icos, Ox40, Gitr, Hvem, CD27, and CD28. In another embodiment, the marker 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 marker may include one or more selected from the group consisting of PD-1, PD-L1, CTLA-4, Lag3, and Tim3. Additionally, the marker may include one or more selected from the group consisting of Icos, Ox40, Gitr, Hvem, CD27, and CD28.

Such a change in the expression level of a marker means a change in a tumor/immune-related marker that affects the tumor-suppressing effect that appears when the anti-CD300c antibody or antigen-binding fragment thereof of the present invention is administered to an individual. For example, the change in the expression level of the marker may include a change in the expression pattern of a protein marker or immune barrier protein marker associated with the activity of immune cells (e.g., dendritic cells, macrophages, T cells, NKT cells), a tumor microenvironment (TME) protein marker that affects tumor growth, and a marker associated with Th1 and Th2 responses. For specific examples of the marker, see the above description. Through such changes in expression pattern, it is possible to predict the probability that a patient will respond to a drug or to select an anticancer drug, including other immune barrier inhibitors, that can maximize the anticancer effect. In addition, by utilizing the marker, it is possible to determine whether a patient can be treated with the antibody. In addition, the effect of drug treatment can be monitored. In addition, information for a treatment method, including drug dosage, dosage, combination therapy, etc., can be provided.

According to an embodiment of the present invention, after administration of the anti-CD300c antibody or antigen-binding fragment thereof of the present invention, it was confirmed that an enhanced antitumor effect was achieved when the antibody was used 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 an individual.

In another embodiment, the method may further include a step of confirming the therapeutic responsiveness of the anti-CD300c antibody or antigen-binding fragment thereof based on the expression levels of the confirmed markers. The markers 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. Also, the markers may include one or more selected from the group consisting of vegfa, pdgfrb, Col4a1, and Hif1a. Also, 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. Additionally, the marker may include one or more selected from the group consisting of Tbx21, Stat1, Stat4, Ifng, Cxcr3, and IL-6.

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 when the expression level of one or more of the markers is decreased 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 when the expression level of one or more markers selected from the group consisting of vegfa, pdgfrb, Col4a1, Hif1a, and IL-6 is decreased compared to a subject not administered with the anti-CD300c antibody or its antigen-binding fragment. In this case, the decrease in the expression level means a statistically significant decrease, and the decrease rate of the 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.

In addition, the method may further include a step of 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 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 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 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.

Pharmaceutical Composition According to another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating cancer, comprising an anti-CD300c antibody or an antigen-binding fragment thereof as an active ingredient.

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

In one embodiment, the pharmaceutical composition may further include 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 included in the same composition or each may be included in a separate composition. When included in separate compositions, the anti-CD300c antibody or antigen-binding fragment thereof and the additional immunological anti-cancer agent may be formulated separately and may be administered simultaneously or sequentially.

To prepare the pharmaceutical composition of the present invention, the antibody or antigen-binding fragment thereof and optionally an additional immunological anticancer agent can be mixed with a pharma- ceutically acceptable carrier and/or excipient. The pharmaceutical composition can be prepared in the form of a lyophilized formulation or an aqueous solution. For example, see Remington's Pharmaceutical Sciences and U.S. Pharmacopeia: National Formulary, Mack Publishing Company, Easton, PA (1984).

Acceptable carriers and/or excipients (including stabilizers) are non-toxic to a subject at the dosages and concentrations employed and include, but are not limited to, buffers (e.g., phosphate, citrate or other organic acids); antioxidants (e.g., ascorbic acid or methionine); preservatives (e.g., octadecyldimethylbenzyl ammonium 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 less residues) polypeptides; proteins (e.g., serum albumin, zeolite, glycerol, sorbitol ... The surfactants may include, but are not limited to, 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); salt-forming counterions (e.g., sodium); metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants (e.g., TWEEN®, PLURONICS®, or polyethylene glycol (PEG)).

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

As used herein, the term "prophylactically or therapeutically effective amount" or "effective amount" refers to an amount of the active ingredient of a composition that is effective in preventing or treating cancer in a subject, and means an amount sufficient to prevent or treat cancer at a reasonable benefit/risk ratio applicable to medical treatment and without causing side effects. The level of the effective amount can be determined based on factors including the patient's health condition, type and severity of disease, drug activity, sensitivity to the drug, administration method, administration time, administration route and excretion rate, treatment period, co-administered or concomitant drugs, and other factors well known in the medical field. In this case, it is important to administer an amount that provides the maximum effect with minimal side effects or minimal amount without side effects, taking all of the above factors into consideration, which can be easily determined by a person of ordinary skill in the art.

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, about 0.01 mg to 100 mg, or 5 mg to about 50 mg per kg of body weight, can be administered once or several times a day. However, since the amount may increase or decrease depending on the route and period of administration, severity of the disease, sex, weight, age, etc., the scope of the present invention is not limited to these amounts.

According to another aspect of the present invention, there is provided a kit for preventing or treating cancer , comprising a composition containing the 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.

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

In one embodiment, the instructions may include instructions including dosing or administration guidelines for the active ingredient. For example, the instructions may include instructions for measuring the expression level of CD300c protein using a biological sample or material obtained from the subject prior to administration of the antibody or antigen-binding fragment thereof. Optionally, the kit may include an instrument or device necessary to administer the active ingredient(s).

Dosage: Effective or non-toxic amounts of each of the anti-CD300c antibody or antigen-binding fragment thereof according to the present invention, and optionally, additional immunological anti-cancer agents, 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 age, sex, medical complications, and weight of the subject, and the ability of the components to elicit the desired response in the subject, and the dosage of the anti-cancer agent used in combination. The dosage and administration regimen of each of the anti-CD300c antibody or antigen-binding fragment thereof or additional immunological anti-cancer agent can be adjusted to provide an optimal therapeutic response. For example, several divided doses can be/can be administered daily, weekly, every two weeks, every three weeks, every four weeks, etc., and the dosage can be proportionally reduced or increased according to the exigencies of the therapeutic situation.

According to another aspect of the present invention, there is provided a method for providing information for the prevention or treatment of cancer , comprising determining an expression level of CD300c protein using a biological sample or data 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 an antigen-binding fragment thereof.

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 mRNA of CD300c, such as a primer pair or a probe. Methods for determining the expression level using these reagents, including those described above, are well known in the art.

In one embodiment, the method may include a step of determining that the subject is suitable for treatment using an anti-CD300c antibody or an antigen-binding fragment thereof when the expression level of CD300c protein determined using a biological sample or material of the subject is at or above a certain level. Specifically, the method may include a step of determining that the subject is suitable for treatment using an anti-CD300c antibody or an antigen-binding fragment thereof when the expression level of CD300c protein determined using a biological sample or material of the subject is statistically significantly higher (e.g., 10% or higher) than a control group (e.g., expression level in normal people without cancer or average expression level in cancer patients). However, the presented differences in the expression level of CD300c protein are merely exemplary and may be 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, and are not limited thereto. Preferably, the control group may be the average expression level in patients with the same type of cancer.

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

In other embodiments, the information for preventing or treating cancer may include, but is not limited to, information regarding one or more of therapeutic responsiveness to a therapeutic agent (e.g., an anti-CD300c antibody or an antigen-binding fragment thereof) associated with CD300c protein, 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 cancer therapeutic responsiveness to an anti-CD300c antibody or an antigen-binding fragment thereof, survival time of a subject, or both.

According to another aspect of the present invention, there is provided 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 material obtained from a subject in need of cancer prevention or treatment. The kit may comprise one or more other component compositions, solutions or devices suitable for an analytical method. The kit may be a kit for measuring the expression level of a protein marker, for example, an enzyme-linked immunosorbent assay (ELISA) kit. The kit may comprise other reagents required for immunological detection of the antibody and known in the art. The kit may further comprise a pharmaceutical composition comprising an anti-CD300c antibody or an antigen-binding fragment thereof as an active ingredient.

The present invention will now be described in more detail with reference to the following examples. However, the following examples are merely intended to illustrate the present invention, and the scope of the present invention is not limited to these examples.

Example I. Preparation of anti-CD300c monoclonal antibody Example 1. Preparation of anti-CD300c monoclonal antibody Example 1.1. Preparation of anti-CD300c monoclonal antibody library To select anti-CD300c monoclonal antibodies, biopanning was performed using lambda phage library, kappa phage library, VH3VL1 phage library, and OPALTL phage library. Specifically, CD300c antigen was added to an immunotube at a concentration of 5 μg/mL and reacted for 1 hour to adsorb the antigen to the surface of the tube. Then, 3% skim milk was added to suppress non-specific reactions, and 10 ¹² PFU of the antibody phage library dispersed in 3% skim milk was added to each immunotube to bind to the antigen. Then, the plate was washed three times with TBST (tris buffered saline-Tween 20) solution to remove non-specifically bound phages, and single-chain variable fragment (scFv) phage antibodies specifically bound to the CD300c antigen were eluted with 100 mM triethylamine solution. The eluted phages were neutralized with 1.0 M Tris-HCl buffer solution (pH 7.8) and then treated with E. coli ER2537 and infected at 37° C. for 1 hour. The infected E. coli was 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, a portion of which was supplemented with 15% glycerol and stored at -80°C until use, and the remaining portion was reinoculated into SB-carbenicillin-2% glucose solution and cultured at 37°C. The resulting culture medium was centrifuged, and biopanning was repeated three times using the supernatant containing phage particles to obtain and concentrate antigen-specific antibodies.

After repeating biopanning three times, the E. coli containing the antibody genes were spread on LB agar medium containing carbenicillin and cultured at 37°C for 16 hours, and the formed E. coli colonies were re-inoculated into SB-carbenicillin-2% glucose solution and cultured at 37°C until the absorbance (OD600nm) reached 0.5, after which IPTG was added and cultured at 30°C for an additional 16 hours. After that, periplasmic extraction was performed, and a library pool of antibodies that specifically bind to the CD300c antigen was initially obtained through the above results.

Example 1.2. Selection of anti-CD300c monoclonal antibodies In order to select anti-CD300c monoclonal antibodies that have high binding affinity and specifically bind 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 in a coating buffer (0.1 M sodium carbonate, pH 9.0) at a concentration of 5 μg/mL per well, and then reacted at room temperature for 3 hours to bind the antigens to the plate. Then, the wells were washed three times with PBST (phosphate buffered saline-Tween 20) to completely remove unbound antigens, and 350 μL of PBST containing 2% BSA (bovine serum albumin) was added to each well, and the wells were incubated at room temperature for 1 hour, and washed again with PBST. Then, 25 μg of plasma membrane extract containing scFv obtained in the same manner as in Example 1.1 was added to each well, and the wells were incubated at room temperature for 1 hour to bind to the antigens. After 1 hour, the wells were washed three times with PBST to remove unbound scFvs, and 4 μg/mL of detection antibody was added and incubated at room temperature for 1 hour again. Then, the unbound detection antibody was removed using PBST, and anti-rabbit IgG conjugated with HRP was added, and incubated at room temperature for 1 hour, and PBST was again used to remove unbound antibody. Next, a 3,3',5,5'-tetramethylbenzidine (TMB) solution was added and reacted for 10 minutes to develop color, after which a 2N sulfuric acid solution was added to terminate the color reaction, and the absorbance was measured at 450 nm to confirm the antibody that specifically binds to the CD300c antigen.

1.3. Confirmation of Anti-CD300c Monoclonal Antibody Sequence The base 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 complementarity-determining regions (CDRs). As a result, 25 types of anti-CD300c monoclonal antibodies each having a different amino acid sequence were obtained. The heavy and light chain variable regions of the 25 types of anti-CD300c monoclonal antibodies are shown in Tables 3 and 4 below.

In each of the drawings referred to in Tables 3 and 4 above, the CDR regions (CDR1, CDR2, and CDR3) are underlined and shown in order (i.e., CDR1 is shown first, then CDR2, and then CDR3). In addition, the CDR regions included in each drawing are shown by sequence numbers as shown in Table 5 below:

As described above, 25 anti-CD300c monoclonal antibodies have been identified that have high binding affinity to the CD300c antigen and can be used to prevent or treat cancers that specifically bind to the antigen.

Example 1.4. Preparation and purification of anti-CD300c monoclonal antibody Using the base sequence of the anti-CD300c monoclonal antibody confirmed in Example 1.3, an expression vector was prepared by separating heavy and light chains capable of expressing the antibody. More specifically, genes were inserted into the pCIW3.3 vector to express the heavy and light chains, respectively, using the analyzed CDR sequence. The prepared heavy and light chain expression vectors were mixed with PEI (polyethyleneimine) in a mass ratio of 1:1 and transfected into 293T cells to induce antibody expression, and on the 8th day, the culture medium was centrifuged to remove the cells, and a culture medium was obtained. The obtained culture medium was filtered and then resuspended using a solution containing 0.1 M NaH ₂ PO ₄ and 0.1 M Na ₂ HPO ₄ (pH 7.0). The resuspended solution was purified by affinity chromatography using protein A beads (GE healthcare) and finally eluted using elution buffer (Thermofisher).

To confirm the produced antibodies, 5 μg of purified antibodies were added to reducing and non-reducing sample buffers, respectively, and electrophoresis was performed using pre-made SDS PAGE (Invitrogen), after which the proteins were stained using Coomassie blue. The results under non-reducing conditions are shown in Figure 4, and the results under reducing conditions in Figure 5.

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

II. Expression of CD300c in Cancer Cells and Binding of Anti-CD300c Monoclonal Antibody to 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 In order to evaluate whether CD300c is expressed in various cancer cells, various cell lines such as cancer cell lines 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) were cultured and the expression of CD300c was evaluated at the mRNA and protein levels. In addition, the immune cell THP-1 (human monocyte cell line) was also evaluated. At this time, HEK293T (general cell line) was used as a control group.

Meanwhile, 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 then blocked with 5% normal bovine serum albumin. Then, the cells were stained with 0.5 μg of eFluor660-labeled anti-CD300c antibody (Invitrogen). Then, the fluorescently labeled cells were confirmed using flow cytometry (FACS).

As a result, it was confirmed that CD300c antigen is expressed at the mRNA and protein levels in various cancer cells, including colon cancer, lung cancer, and breast cancer. In addition, as shown in Figure 6, analysis using flow cytometry (FACS) confirmed that CD300c was expressed in significantly higher amounts in human lung cancer cell line (A549) and human monocyte cell line (THP-1) compared to a general cell line (HEK293T).

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 colon cancer patient's tissue was fixed in formalin and cut into blocks using paraffin, then sliced into 2.0 mm diameter and 3-5 um thick slices for a microtissue array. The slices were then attached to a slide in a certain direction and dried. The cancer tissue was stained using H&E staining, and then treated with anti-CD300c antibody (Invitrogen) at a dilution of 1:500 to stain CD300c. As a result, as shown in Figure 7a, it was confirmed that CD300c was expressed in the patient's colon cancer tissue.

To confirm whether CD300c is expressed not only in colon cancer patient tissues but also in immune cells within cancer tissues, ^2X105 CT26 cells were subcutaneously injected into 8-week-old BALB/c mice. Tumor tissues were collected from 6 control mice that were sacrificed on the 25th day (D25) after tumor implantation and did not receive anti-CD300c antibody. The tumor tissues were removed and incubated at 37°C for 1 hour in a mixture of collagenase D (20mg/ml) and DNase I (2mg/ml). The tissues were then filtered through a 70μm cell strainer, red blood cells were dissolved, and the tissues were refiltered through a nylon mesh. The single cell suspension was then blocked with CD16/32 antibody (obtained from Invitrogen) and cells were stained with cell viability stain (obtained from Invitrogen) and antibodies against total macrophage markers F4/80 (Abeam), CD11b (obtained from Abcam), CD11c (obtained from Abcam), CD3 (obtained from Abcam), CD4 (obtained from Thermofisher), CD8 (obtained from Thermofisher) and CD300c antibody (obtained from Sino Biological). Data were then read on a CytoFLEX flow cytometer and analyzed with FlowJo software.

As a result, as shown in Figure 7b, it was confirmed that immune cells expressing CD300c were present in the mouse tumor tissue. These immune cells 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.

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 tonsil tissues and tissues from colon cancer patients were fixed in formalin and blocks were made with paraffin. Then, the normal tonsil tissues and tissues from colon cancer patients were searched for positions for tissue microarrays, and the tissues were sliced into 2.0 mm diameter and 3-5 um thick slices for microtissue arrays. The slices were then attached to slides in a certain direction and dried. The cancer tissues were stained with H&E staining, and then treated with anti-CD300c antibody (Invitrogen) at 1:500 to stain CD300c.

As a result, as shown in Figures 8a and 8b, it was confirmed that CD300c was expressed in both normal tonsillar tissue (Figure 8a), which is an immune tissue, and in the patient's colon cancer tissue (Figure 8b). Since many immune cells such as T cells and monocytes are distributed in the tonsils, the expression of CD300c in tonsillar tissue means that CD300c is expressed in immune cells. This experimental example, like Experimental Example 1.2, confirmed the expression of CD300c in the tissues of colon cancer patients, but by observing the expression of CD300c in all four colon cancer patient tissues, it means that it was confirmed that CD300c is expressed in many colon cancer tissues.

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 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 a coating buffer solution (0.1 M sodium carbonate, pH 9.0) at a concentration of 8 ug/mL per well onto an ELISA plate, and the antigen was allowed to bind to the plate by reacting at room temperature for 3 hours. Then, after washing three times with PBST to completely remove unbound antigens, 300 μL of PBST containing 5% BSA (bovine serum albumin) was added to each well, and the wells were incubated at room temperature for 1 hour, and washed again with PBST. Then, anti-CD300c monoclonal antibody was diluted 4-fold and added, and incubated at room temperature for 1 hour to bind to the antigen. After 1 hour, the wells were washed three times with PBST to remove unbound anti-CD300c monoclonal antibody, and 4 μg/mL of detection antibody (HRP conjugated anti-Fc IgG) was added, and incubated at room temperature for 1 hour again. Then, after removing unbound detection antibody with PBST, TMB solution was added, and the incubation was continued for 10 minutes to develop color, and 2N sulfuric acid solution was added to terminate the color development reaction, and the absorbance was measured at 450 nm to confirm the antibody that specifically binds to the CD300c antigen. The results are shown in Table 6 and FIG. 9.

As shown in Table 6, the EC50 (The effective concentration of drug that causes 50% of the maximum response) of the anti-CD300c monoclonal antibodies was measured, and the binding affinity of the remaining 14 clones, except for 4 clones (CK3, CL8, SK15, and SK16), was 0.2 μg/mL or less, confirming that the binding affinity was high. In addition, as shown in FIG. 9, the sigmoid curve of the binding ELISA results also confirmed that the anti-CD300c monoclonal antibody of the present invention binds to the CD300c antigen with strong binding affinity.

Experimental Example 2.2. Confirmation of Cellular Antigen Recognition by Anti-CD300c Monoclonal Antibody To confirm that anti-CD300c monoclonal antibody (CL7) recognizes cellular antigens, FACS binding was performed.

After overexpressing CD300c in 293T cells (ATCC) and THP-1 cells (ATCC), the cells were dispensed at ^2x105 cells per microcentrifuge tube. Then, anti-CD300c monoclonal antibody, which was serially diluted 3-fold from 10ug/ml, was reacted in a _CO2 incubator for 30 minutes, and washed twice with FACS buffer. Then, FITC-conjugated anti-human IgG (H+L) diluted 1:100 in FACS buffer was reacted in a _CO2 incubator for 30 minutes, and washed twice with FACS buffer. Then, FITC signals were measured using a Beckman Coulter CytoFLEX instrument, and MFI values were calculated using the CytExpert program. Using the MFI values thus obtained, a sigmoidal curve was drawn using the sigmaplot program to calculate the EC50 (The effective concentration of drug that causes 50% of the maximum response). As a result, the EC50 was 2.7 nM for 293T cells and 2.6 nM for THP-1 cells.

As shown in FIG. 10, in the sigmoidal curve of the FACS binding results, the anti-CD300c monoclonal antibody prepared in Example 1 bound to CD300c overexpressed on the surface of THP-1 and 293T cells with strong binding force. Therefore, it was confirmed that the anti-CD300c monoclonal antibody binds to CD300c antigen-specifically.

Experimental Example 2.3. Confirmation of binding ability of anti-CD300c monoclonal antibody to CD300c antigen (I): Binding ELISA
CD300c antigen (250ug/mL) was diluted to a concentration of 800ng/mL in coating buffer solution (0.1M sodium carbonate, pH 9.0) and 100uL was added to a 96-well microplate and incubated overnight at 4°C. The next day, the plate was washed three times with 200uL of PBST. Then, 200uL of blocking buffer solution (5% skim milk) was added to each well and blocked at room temperature for 1 hour. CL7, an anti-CD300c monoclonal antibody, was diluted to 200ug/mL in PBS and measured with Nanodrop (Nanodrop One/One ^c , manufacturer: Thermo Fisher Scientific) to confirm the concentration. Then, CL7 was diluted 4-fold from 10ug/mL in PBS and 100uL was added to each well and incubated at room temperature for 1 hour. After the reaction, the plate was washed three times with 200 uL of PBST. 100 uL of a secondary antibody (conjugated anti-Fc IgG) was added to the blocking buffer solution at a ratio of 1:10,000, and the plate was then reacted at room temperature for 1 hour. Then, 200 uL of PBST was added, and the plate was washed three times. Then, 100 uL of TMB and hydrogen peroxide were mixed at a ratio of 1:1 and added to each well, and the plate was then reacted at room temperature for 7 to 9 minutes. Then, 50 uL of 1N sulfuric acid was added to stop the color development, and the binding affinity was measured at 450 nm using a microplate reader (product name: Varioskan LUX) to obtain the binding affinity results.

As a result, as shown in FIG. 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 strength and specificity for the antigen CD300c.

Experimental Example 2.4. Confirmation of binding ability 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.

To immobilize CD300c on a CM5 chip, 5ug/ml of CD300c was diluted in 10mM acetate buffer (pH 5.5). Then, all flow rates were set to 10ml/min, and the target RU for each was set to 300RU. Activation was performed with a mixture of 0.2M EDC and 0.05M NHS, blocked with 1M ethanolamine, and immobilized to a final RU of 399.2RU for CD300c. Then, CL7 was diluted in PBSP to concentrations of 0, 0.195, 0.39, 0.78, 1.56, 3.125, and 6.25ug/ml, and a kinetics/affinity test was performed with a binding time of 240 seconds, a dissociation time of 900 seconds, and a flow rate of 30ul/min. The surface was then regenerated by flowing 50 mM NaOH at a rate of 30 ul/min for 30 seconds.

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. This indicates that the binding strength of the anti-CD300c monoclonal antibody to the antigen is high.

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 does not bind to other B7 family proteins but specifically binds only to CD300c, a binding ELISA was performed. More specifically, CD300c antigen, CD300a antigen, or seven types of 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), CD273 [PD-L2] (Sino Biological)) were coated on an ELISA plate in a coating buffer solution (0.1 M sodium carbonate, pH 9.0) to a concentration of 8 μg/mL per well, and then the plate was incubated overnight at 2° C. to 8° C. to allow the antigens to bind to the plate. After washing three times with PBST to completely remove unbound antigens, 300 ml of blocking buffer solution (5% skim milk in PBST) was added to each well. Then, after blocking at room temperature for 1 hour, the wells were washed again with PBST. Then, CL7 was diluted 4-fold in PBS and reacted at room temperature for 1 hour to bind to the antigen. After 1 hour, the wells were washed three times with PBST. Then, a secondary antibody (HRP-conjugated anti-Fc IgG) diluted at 4 μg/mL in blocking buffer solution was added and reacted at room temperature for 1 hour again. Then, unbound detection antibodies were removed using PBST, and TMB solution was added and reacted for 10 minutes to develop color. Then, 2N sulfuric acid solution was added to terminate the color development reaction, and the absorbance was measured at 450 nm to confirm the antibody that specifically binds to the CD300c antigen.

As a result, as shown in Figure 13, it was confirmed that the anti-CD300c monoclonal antibody does not bind to other similar proteins and specifically recognizes only CD300c.

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 to the CD300c antigen, we further confirmed whether CL7 exhibits cross-reactivity to the CD300a antigen, which has a similar protein sequence to that of the CD300c antigen, in addition to the fact that CL7 is already known to have an antagonistic effect on the CD300c antigen. More specifically, we treated the cells with CD300a antigen (obtained from Sino Biological) at concentrations of 0.039, 0.63, and 10 μg/mL, and then performed binding ELISA in the same manner as in Experimental Example 2.1.

As a result, as shown in Figure 14, it was confirmed that the anti-CD300c monoclonal antibody does not bind to any antigens other than CD300c, and therefore exhibits high binding specificity only to the CD300c antigen.

Experimental Example 2.7. Comparison of overall survival of various cancer patients according to CD300c expression level Using data of kidney cancer (530 patients), pancreatic cancer (177 patients), and liver cancer (370 patients) patients obtained from the US TCGA (The Cancer Genome Atlas) database, overall survival according to the expression level of CD300c was compared. First, each cancer patient was classified according to the level of CD300c expression. Such high and low were classified by comparing with the average expression level of CD300c by cancer type. In the case of kidney cancer patients, 394 patients showed low CD300c expression level, and 136 patients showed high CD300c expression level. In the case of pancreatic cancer patients, 57 patients showed low CD300c expression level, and 120 patients showed high CD300c expression level. Meanwhile, 192 liver cancer patients showed low CD300c expression levels, while 178 showed high CD300c expression levels. The overall survival time according to the CD300c expression level of each cancer patient was analyzed using the Kaplan-Meier method. The survival time of patients with high and low CD300c expression levels was then compared using the log-rank test.

As a result, as shown in FIG. 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 mean that CD300c expression is highly correlated with the survival time of cancer patients, but also that suppressing the expression or activity of CD300c can be expected to have a therapeutic effect on cancer or increase survival time.

III. Anti-cancer effect of anti-CD300c monoclonal antibody 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 In order 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 by treatment of human T cells with anti-CD300c monoclonal antibody was confirmed. IL-2 is an immune factor that aids in the growth, proliferation, and differentiation of T cells, and an increase in the amount of IL-2 produced means that the stimulation that induces increased differentiation, proliferation, and growth of T cells is increased, 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, and then immobilized for 24 hours. Jurkat T cells (human T lymphocyte cell line) were treated with 1×10 ⁵ cells/well of anti-CD300c monoclonal antibody at 10 μg/well. The amount of IL-2 produced was then 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 FIG. 16.

As shown in FIG. 16, it was confirmed that the amount of IL-2 produced increased when anti-CD300c monoclonal antibody was treated with Jurkat T cells activated by treatment with anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody. From the above results, it was confirmed that anti-CD300c monoclonal antibody activates T cells, thereby inducing anti-cancer immunity and suppressing the growth of cancer tissue.

Experimental Example 3.2. Confirmation of Promotion of Differentiation into M1 Macrophages (I): Measurement of the Amount of Macrophage Differentiation Marker (TNF-α) Produced In order to confirm whether the anti-CD300c monoclonal antibody selected in Example 1 can promote the differentiation of monocytes into M1 macrophages, THP-1 (human monocyte cell line) was dispensed at 1.5×10 ⁴ cells/well into a 96-well plate and treated with 10 μg/mL of anti-CD300c monoclonal antibody and/or 100 ng/mL of LPS. After reacting for 48 hours, the amount of TNF-α (Tumor necrosis factor-α), a differentiation marker of M1 macrophages, produced was measured using an ELISA kit (Human TNF-α Quantikine kit, R&D Systems). The results are shown in FIG. 17 and FIG. 18.

As shown in Figure 17, it was confirmed that the CL4, CL7, CL10, and SL18 anti-CD300c monoclonal antibodies increased the amount of TNF-α produced by approximately two-fold or more compared to the control group (Con) treated with LPS alone.

In addition, as shown in FIG. 18, it was confirmed that the amount of TNF-α produced was increased in all experimental groups treated with anti-CD300c monoclonal antibody alone without LPS treatment, compared to the control group (Con) treated with LPS alone.

Experimental Example 3.3. Confirmation of antibody concentration-dependent increase in differentiation ability into M1 macrophages To confirm that the ability of anti-CD300c monoclonal antibody to induce differentiation into M1 macrophages increases depending on the concentration, the amount of TNF-α produced was confirmed in the same manner as in Experimental Example 3.2. Anti-CD300c monoclonal antibody was treated at concentrations of 10, 1, and 0.1 μg/mL. The results are shown in FIG. 19. As shown in FIG. 19, it was confirmed that the amount of TNF-α produced increased as the treatment concentration of anti-CD300c monoclonal antibody (CL7, CL10, or SL18) increased.

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.

Experimental Example 3.4. Confirmation of promotion of differentiation into M1 macrophages (II): Observation of cell morphology In order to confirm the differentiation into M1 macrophages by cell morphology when monocytes were treated with anti-CD300c monoclonal antibody, THP-1 was 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 FIG.

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. From the above results, it was confirmed that treatment with anti-CD300c monoclonal antibody promotes the differentiation of monocytes into M1 macrophages.

Experimental Example 3.5. Reconfirmation of promotion of differentiation into M1 macrophages To reconfirm whether CL7 anti-CD300c monoclonal antibody promotes 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 an ELISA kit. More specifically, THP-1 was dispensed into a 96-well plate at 1.5×10 ⁴ cells/well and treated with 10 μg/mL anti-CD300c monoclonal antibody. After reacting for 48 hours, the production levels of TNF-α, IL-1β, and IL-8, which are differentiation markers of M1 macrophages, were measured using an ELISA kit (Human TNF-α Quantikine kit, R&D Systems). The results are shown in FIG. 22.

As shown in Figure 22, it was confirmed that all three M1 macrophage differentiation markers were 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.

Experimental Example 3.6. Confirmation of redifferentiation ability of M2 macrophages to M1 macrophages In order to confirm whether anti-CD300c monoclonal antibody can redifferentiate M2 macrophages to ^M1 macrophages, THP-1 was dispensed at 1.5x104 cells/well into a 96-well plate, pretreated with 320nM PMA for 6 hours, and then treated with 20ng/mL IL-4 (Interleukin-4) and IL-13 (Interleukin-13), and 10μg/mL anti-CD300c monoclonal antibody, and reacted for 18 hours. The production amounts of TNF-α, IL-1β, and IL-8 were confirmed by ELISA kit. The results are shown in Figures 23 to 25.

As shown in Figures 23 to 25, when the cells were not pretreated with PMA, the production of TNF-α, IL-1β, and IL-8 increased in the experimental group treated with IL-4, IL-13, and anti-CD300c monoclonal antibody, and when the cells were pretreated with PMA, the production of TNF-α, IL-1β, and IL-8 increased in the experimental group treated with IL-4, IL-13, and anti-CD300c monoclonal antibody. From these results, it was confirmed that anti-CD300c monoclonal antibody effectively redifferentiates M2 macrophages back into M1 macrophages.

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 into M1 macrophages, THP-1 was dispensed at ^1.5x104 cells/well into a 96-well plate, pretreated with 10 μg/mL anti-CD300c monoclonal antibody for 48 hours, and then treated with 100 ng/mL PMA, 100 ng/mL LPS, and 20 ng/mL IL-4 and IL-13 for 24 hours. The amount of TNF-α produced was confirmed using an ELISA kit. The results are shown in FIG. 26.

As shown in Figure 26, it was confirmed that the amount of TNF-α produced was significantly increased in all experimental groups pretreated with anti-CD300c monoclonal antibody compared to 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. From the above results, it was confirmed that anti-CD300c monoclonal antibody has excellent differentiation ability of M0 macrophages into M1 macrophages, differentiation ability of THP-1 into M1 macrophages, and re-differentiation ability of M2 macrophages into M1 macrophages.

Experimental Example 4. Confirmation of interspecies cross-reactivity of anti-CD300c monoclonal antibody by observing anti-cancer effect Experimental Example 4.1. Confirmation of growth inhibitory effect on human cancer cells In order to confirm the effect of monoclonal antibody targeting CD300c on the growth of cancer cells, cell proliferation analysis was performed using A549 (human lung cancer cell line). More specifically, ^2x104 cells were dispensed in a 96-well plate under the condition of 0% fetal bovine serum (FBS), and ^6x103 cells were dispensed under the condition of 0.1% fetal bovine serum. Then, 10μg/mL of anti-CD300c monoclonal antibody was treated and cultured for 5 days. The cancer cell growth inhibitory effect of anti-CD300c monoclonal antibody was confirmed by treating with CCK-8 (DOJINDO) and measuring absorbance at OD450nm. The results are shown in Figures 27 and 28.

As shown in Figure 27, it was confirmed that all of the compounds except for SK11 and SK17 had the effect of suppressing the proliferation of cancer cells under 0% FBS conditions.

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

Experimental Example 4.2. Confirmation of the cancer cell growth inhibitory effect according to the concentration of anti-CD300c monoclonal antibody To confirm the cancer cell growth inhibitory effect according to the concentration of anti-CD300c monoclonal antibody, ^2x104 A549 cells were dispensed into a 96-well plate under the condition of 0% fetal bovine serum (FBS), treated with 10μg/mL anti-CD300c monoclonal antibody, and cultured for 5 days. Then, the cells were treated with CCK-8 (DOJINDO) and reacted for 3 hours, and the absorbance was measured at OD450nm to confirm the cancer cell growth inhibitory effect of anti-CD300c monoclonal antibody. The results are shown in Figure 29.

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

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 dispensed into a 96-well plate at a concentration of ^1x104 cells/well, and then treated with 10 μg/mL of anti-CD300c monoclonal antibody and cultured. The amount of TNF-α produced was confirmed using an ELISA kit. The results are shown in Figure 30.

As shown in Figure 30, the amount of TNF-α produced was increased in the experimental group treated with anti-CD300c monoclonal antibody. This result indicates that anti-CD300c monoclonal antibody has cross-reactivity that acts similarly not only in humans but also in mice to promote differentiation into M1 macrophages.

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 have an anti-cancer effect, CT26 (mouse colon cancer cell line) was dispensed into a 96-well plate at a concentration of ^1x104 cells/well, treated with 10ug/mL of monoclonal antibody, and cultured for 5 days, and then cell proliferation analysis was performed using CCK-8 detection.

As shown in Figure 31, the anti-CD300c monoclonal antibody was confirmed to have a cancer treatment effect in mice, as it exerted a cancer cell proliferation inhibitory effect of 66% (CL7), 15% (CL10), and 38% (SL18) compared to the control group. This shows that the anti-CD300c monoclonal antibody has cross-reactivity that acts similarly in mice as well as humans to exert an anti-cancer effect.

Experimental Example 5. Comparison of in vitro anticancer effects between anti-CD300c monoclonal antibody and existing immunological anticancer agents. The immunological anticancer agents used in the following experimental examples were obtained from the following sources: Imfinzi (AstraZeneca) and Keytruda (Merck Sharp & Dohme).

Experimental Example 5.1. Comparison of differentiation ability into M1 macrophages between anti-CD300c monoclonal antibody and existing immunological anticancer drug: Measurement of production amount of three differentiation markers (TNF-α, IL-1β, and IL-8) For comparison, the production amount of TNF-α was confirmed by ELISA kit in the same manner as in Experimental Example 3.2. As an existing immunological anticancer drug, Imfinzi was treated at a concentration of 10 μg/mL. The results are shown in Figure 32.

As shown in Figure 32, it was confirmed that anti-CD300c monoclonal antibody significantly increased the amount of TNF-α produced compared to the comparison group treated with Imfinzi (Imf) alone. From the above results, it was confirmed that anti-CD300c monoclonal antibody significantly increased the differentiation ability into M1 macrophages compared to known immune anticancer drugs.

For comparison with other immune anticancer drugs, the cells were treated with Imfinzi, an anti-PD-L1 immune anticancer drug, Keytruda, an anti-PD-1 immune anticancer drug, and an isotype control (immunoglobulin G) antibody at a concentration of 10 μg/mL, and the amounts of TNF-α, IL-1β, and IL-8 produced were confirmed using an ELISA kit. The results are shown in Figures 33 to 35.

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

Experimental Example 5.2. Comparison of the differentiation ability of M0 macrophages to M1 macrophages between anti-CD300c monoclonal antibody and existing immunological anticancer drug To compare the differentiation ability of M0 macrophages to M1 macrophages between anti-CD300c monoclonal antibody and existing immunological anticancer drug, THP-1 was dispensed at ^1.5x104 cells/well into a 96-well plate and treated with 10μg/mL anti-CD300c monoclonal antibody, 10μg/mL Imfinzi, and/or 200nM PMA (phorbol-12-myristate-13-acetate). After reacting for 48 hours, the amount of TNF-α produced was measured using an ELISA kit. The results are shown in FIG. 36.

As shown in Figure 36, TNF-α was not produced in the comparison group treated with the immunological anti-cancer drug Imfinzi alone, but the amount of TNF-α produced increased in the experimental group treated with anti-CD300c monoclonal antibody alone. In addition, when THP-1 cells were treated with PMA to differentiate into M0 macrophages, the experimental group treated with anti-CD300c monoclonal antibody showed a significantly higher amount of TNF-α produced compared to the experimental group treated with Imfinzi. From the above results, it was confirmed that anti-CD300c monoclonal antibody promotes the differentiation of M0 macrophages into M1 macrophages compared to existing immunological anti-cancer drugs.

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

As shown in Figure 37, when monocytes were differentiated into M1 macrophages by treatment with LPS, the experimental group treated with both Imfinzi and LPS showed no significant difference in the amount of TNF-α produced, but the experimental group treated with both anti-CD300c monoclonal antibody and LPS showed a significant increase in the amount of TNF-α produced compared to the experimental group treated with anti-CD300c monoclonal antibody alone.

Experimental Example 5.4. Comparison of Cancer Cell Growth Inhibitory Effects Between Anti-CD300c Monoclonal Antibody and Existing Immuno-Anti-Cancer Agents In order to compare the cancer cell growth inhibitory effects between anti-CD300c monoclonal antibodies and existing immuno-anti-cancer agents, cell growth inhibitory effects were confirmed using A549 (human lung cancer cell line) and MDA-MB-231 (human breast cancer cell line). More specifically, ^2x104 cells were dispensed in a 96-well plate under the condition of 0% fetal bovine serum (FBS), and ^6x103 cells were dispensed in a condition of 0.1% fetal bovine serum. Then, the cells were treated with 10μg/mL anti-CD300c monoclonal antibody, cultured for 5 days, and observed under an optical microscope. The results are shown in Figures 38 and 39.

As shown in Figure 38, it was confirmed that anti-CD300c monoclonal antibody inhibited the proliferation of cancer cells more effectively than Imfinzi, an immunosuppressant.

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

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 anti-CD300c monoclonal antibody CL7 on tumor-associated macrophages in vivo, ^2x105 colon cancer cell lines (CT26) were subcutaneously implanted into 8-week-old BALB/c mice to prepare a syngeneic mouse tumor model. All animal breeding and experiments were carried out in a specific pathogen free (SPF) facility. Twelve days after transplantation of colon cancer cell lines, anti-CD300c monoclonal antibodies were administered to mice with tumors measuring 50-100 ^mm3 , and the same amount of phosphate buffered saline (PBS) was injected as a control. Mice were intraperitoneally injected at a dose of 25 mg/kg twice a week for two weeks, a total of four times. Mice were sacrificed 25 days after injection, and tumor tissues were collected from six mice in each of the CL7 25 mg/kg administration groups, which showed the highest antitumor effect compared to the control group. The tumor tissues were removed and incubated in a mixed solution of collagenase D (20 mg/ml) and DNase I (2 mg/ml) at 37°C for 1 hour. The cells were then filtered through a 70 μm cell strainer, red blood cells were lysed, and then refiltered through a nylon mesh. The single cell suspension was then blocked with CD16/32 (Invitrogen) antibody, and the 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 (Abcam). Data was then read using a CytoFLEX flow cytometer and analyzed using FlowJo software.

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

Experimental Example 6.2. Confirmation of Increase in Cytotoxic T Cells In order to confirm the effect of anti-CD300c monoclonal antibody CL7 on CD8+ T cells under in vivo conditions, an allograft mouse tumor model was prepared and administered at the same concentration as in Experimental Example 6.1. The mice were sacrificed 25 days after injection, and tumor tissues were collected from six mice in each of the CL7 25 mg/kg administration groups, which had the highest antitumor effect compared to the control group. The tumor tissues were removed and incubated in a mixed solution of collagenase D (20 mg/ml) and DNase I (2 mg/ml) at 37°C for 1 hour. The tissues were then filtered through a 70 μm cell strainer, red blood cells were dissolved, and the tissues were filtered again through a nylon mesh. The single cell suspension was then blocked with CD16/32 (Invitrogen) antibody, and cells were stained with a cell viability stain and CD8+ (Abcam) and CD4+ (Abcam) antibodies. Data were then read on a CytoFLEX flow cytometer and analyzed with FlowJo software.

As a result, as shown in Figure 41, it was confirmed that the number of intratumoral CD8+ T cells increased when anti-CD300c monoclonal antibody was administered alone. This means that administration of anti-CD300c monoclonal antibody increases intratumoral cytotoxic T cells, resulting in a cancer treatment effect.

Experimental Example 6.3. Confirmation of Tumor-Specific Increase of Cytotoxic T Cells To confirm whether anti-CD300c monoclonal antibody CL7 increases the number of CD8+ T cells in a tumor-specific manner, an allograft mouse tumor model was prepared and administered at the same concentration as in Experimental Example 6.1. The mice were sacrificed 25 days after injection, and tumor tissues were collected from six mice in each of the CL7 25 mg/kg administration group, which had the highest antitumor effect compared to the control group. The tumor tissues were removed and incubated at 37° C. for 1 hour in a mixed solution of collagenase D (20 mg/ml) and DNase I (2 mg/ml). The tissues were then filtered through a 70 μm cell strainer, red blood cells were lysed, and the tissues were filtered again through a nylon mesh. The single cell suspension was then blocked with CD16/32 (obtained from Invitrogen) antibody, and the cells were stained with a cell viability stain and AH1 tetramer antibody (obtained from Abcam). Data was then read on a CytoFLEX flow cytometer and analyzed with FlowJo software.

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 factor, increased in CD8+ T cells, and the number of CD8+ T cells increased in a tumor (CT26)-specific manner. This means that administration of anti-CD300c monoclonal antibody increased CD8+ T cells to target and suppress CT26 cancer cells.

Experimental Example 6.4. Confirmation of Increased Activity of Cytotoxic T Cells In order to confirm the effect of anti-CD300c monoclonal antibody CL7 on CD8+ T cells under in vivo conditions, an allograft mouse tumor model was prepared and administered at the same concentration as in Experimental Example 6.1. The mice were sacrificed 25 days after injection, and spleens were taken from six mice in each of the CL7 25 mg/kg administration group, which had the highest antitumor effect compared to the control group. IFN-g was then measured using ELISPOT analysis to confirm the results. Specifically, a Mouse IFN-g ELISpot kit from R&D Systems (#EL485) was purchased, and IFN-g was measured according to the kit's protocol.

As a result, as shown in Figure 43, it was confirmed that the expression of IFN-g increased when anti-CD300C monoclonal antibody was administered alone. This means 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 exerting a cancer treatment effect.

Experimental Example 6.5. Confirmation of Increase in Cytotoxic T Cells Compared to Regulatory T Cells In order to confirm the effect of anti-CD300c monoclonal antibody CL7 on the increase in cytotoxic T cells compared to regulatory T cells under in vivo conditions, an allograft mouse tumor model was prepared and administered at the same concentration as in Experimental Example 6.1. The mice were sacrificed 25 days after injection, and tumor tissues were collected from six mice in each of the CL7 25 mg/kg administration group, which had the highest antitumor effect compared to the control group. The tumor tissues were removed and incubated in a mixed solution of collagenase D (20 mg/ml) and DNase I (2 mg/ml) at 37° C. for 1 hour. The tissues were then filtered through a 70 μm cell strainer, red blood cells were lysed, and the tissues were filtered again through a nylon mesh. The single cell suspension was then blocked with CD16/32 (Invitrogen) antibody, and the cells were 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.

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. This means that the CD8+ T cells, the number of which increased with the administration of anti-CD300C monoclonal antibody, further suppress the growth of cancer.

Experimental Example 6.6. Confirmation of the effect on cytotoxic T cells, regulatory T cells, and tumor-associated macrophages In order 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 performed. An allograft mouse tumor model was prepared in the same manner as in Experimental Example 6.1. Colon cancer cell lines were transplanted, and 12 days later, anti-CD300c monoclonal antibodies were administered to mice with tumors measuring 50-100 ^mm3 , and the same amount of phosphate buffered saline (PBS) was injected as a control group. Mice were intraperitoneally injected at a dose of 25 mg/kg twice a week for two weeks, a total of four times. The mice were sacrificed 25 days after injection, and tumor tissues were collected from six mice in each of the CL7 25 mg/kg administration groups, which had the highest antitumor effect compared to the control group. After removing the tumor tissue, it was incubated in a mixed solution of collagenase D (20 mg/ml) and DNase I (2 mg/ml) at 37° C. for 1 hour. Then, it was filtered through a 70 μm cell strainer, red blood cells were lysed, and then it was filtered again through a nylon mesh. Then, the single cell suspension was blocked with CD16/32 (obtained from Invitrogen) antibody, and the cells were stained with a staining solution for confirming cell viability, and CD8+ antibody and CD4+ antibody, which are CD8+ T cell markers, or Foxp3 antibody and CD4+ antibody, which are Treg cell markers, or with F4/80, a marker for total macrophages, and an antibody against iNOS, an M1 macrophage marker (obtained from Abcam). Then, the data was read using a CytoFLEX flow cytometer and analyzed using FlowJo software. The results are shown in FIG. 45.

As shown in Figure 45, anti-CD300c monoclonal antibody (CL7) was confirmed to significantly increase activated CD8+ T cells, suppress regulatory T cells, and repolarize tumor-associated macrophages toward the M1 phenotype.

Experimental Example 6.7. Confirmation of in vivo cancer growth suppression effect In order to confirm the anti-cancer effect of anti-CD300c monoclonal antibody CL7 under in vivo conditions, ^2x105 colon cancer cell lines (CT26) were subcutaneously implanted into 8-week-old BALB/c mice to prepare homograft mouse tumor models. All animal breeding and experiments were carried out in an SPF facility. On the 11th day (D11) after implantation of the colon cancer cell lines, anti-CD300c monoclonal antibodies were administered at 1 mg/kg, 5 mg/kg, 10 mg/kg, or 25 mg/kg to mice with tumors measuring 50-100 ^mm3 , and the control group was injected with the same amount of phosphate buffered saline (PBS). Specifically, the mice were intraperitoneally injected with each dose twice a week for a total of four times over two weeks (D11, D14, D18, and D21). The tumor volume was measured for 25 days. The results are shown in Figure 46.

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

IV. Changes in Biomarker Expression by Administration of Anti-CD300c Monoclonal Antibody Example 2. Changes in Expression of Immune Cell-Related Markers and Tumor Microenvironment-Related Markers by Administration of Anti-CD300c Monoclonal Antibody Example 2.1. Nanostring Immune Profiling In order to confirm changes in expression of immune cells and tumor microenvironment-related markers when the anti-CD300c monoclonal antibody (CL7) prepared in Example ¹ was administered to a solid tumor model, 2x105 colon cancer cell lines (CT26) were subcutaneously implanted into 8-week-old BALB/c mice to prepare an allograft mouse tumor model. All animal breeding and experiments were carried out in an SPF facility. Twelve days after transplanting a colon cancer cell line, anti-CD300c monoclonal antibodies were administered to mice with tumors measuring 50-100 ^mm3 , and the same amount of phosphate buffered saline (PBS) was injected into the control group. The mice were intraperitoneally injected with 25 mg/kg twice a week for a total of four times over two weeks. The mice were euthanized 25 days after injection to prepare tumor tissues. RNA was extracted and purified from the tissues, and changes in dendritic cell markers, macrophage markers, tumor microenvironment (TME) markers, Th1 response markers, or Th2 response markers were confirmed through nanostring immune profiling.

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

In addition, the changes in dendritic cell markers, macrophage markers, tumor microenvironment markers, Th1 response markers, and Th2 response markers following administration of anti-CD300c monoclonal antibody were observed compared to the control group, and the results are shown in Figure 48. As shown in Figure 48, it was confirmed that 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 help cancer growth in the tumor microenvironment; and increased the expression of markers that can confirm Th1 response, Tbx21, Stat1, Stat4, Ifn-g, and Cxcr3.

Example 2.2. Changes in Expression of Immune Barrier Markers Based on the Nanostring immune profiling results obtained in Example 2.1, we identified 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.

The results are shown in Figure 49. It was confirmed that when anti-CD300c monoclonal antibody was administered, the expression of PD-1, CTLA-4, and Lag3 of the inhibitory immune barrier (inhibitory IC) increased, and the expression of ICOS, OX40, Gitr, Cd27, and Cd28 of the agonistic immune barrier (agonistic IC) also increased.

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

V. Combined Use of Anti-CD300c Monoclonal Antibody and Anticancer Immunotherapy Example 3 Combined Administration of Anti-CD300c Monoclonal Antibody (CL7) and Anticancer Immunotherapy The anti-CD300c monoclonal antibody (CL7) produced in Example 1 was combined with other anticancer immunotherapy agents, for example, anti-PD-L1 antibodies Imfinzi(R) and Opdivo(R), anti-PD-1 antibodies Keytruda, anti-CD47 antibodies (αCD47), and anti-CTLA-4 antibodies, and the results were observed.

The sources of the above immunotherapy anticancer drugs are as follows: Imfinzi (AstraZeneca); Opdivo, an anti-CTLA-4 antibody (Bristol Myers Squibb Company), Keytruda (Merck Sharp & Dohme), and an anti-CD47 antibody (Abcam).

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

As a result, as shown in FIG. 50, it was confirmed that the morphology of THP-1 cells changed from suspension cells to round adherent cells, which are the morphology of M1 macrophages, when treated in combination with anti-CD300c monoclonal antibody compared to treatment with the immunological anticancer drug alone. From the above results, it was confirmed that the combination treatment of anti-CD300c monoclonal antibody and immunological anticancer drug further promoted the differentiation of monocytes into M1 macrophages.

Experimental Example 7.2. Confirmation of increase in M1 macrophage markers In order to confirm whether the induction of differentiation of monocytes into M1 macrophages is increased when CL7, an anti-CD300c monoclonal antibody, is treated 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, THP-1 was dispensed into a 96-well plate at 1.5 x ¹⁰⁴ cells/well, and treated with the anti-CD300c monoclonal antibody and the anti-cancer agent at 10 μg/mL, either alone or in combination. After 48 hours of reaction, the production amounts of TNF-α (Tumor necrosis factor-α), IL-1b, and IL-8, which are differentiation markers of M1 macrophages, were measured using an ELISA kit (Human TNF-α Quantikine kit, R&D Systems).

As a result, as shown in FIG. 51, when anti-CD300c monoclonal antibody was used alone, the production of all three differentiation markers increased, and in particular, the production of IL-8 was significantly increased. Also, as shown in FIG. 52, it was confirmed that the production of TNF-α, a marker for M1 macrophages, was further increased when anti-CD300c monoclonal antibody was used in combination with Imfinzi, Opdivo, Keytruda, and αCD47, compared to when anti-CD300c monoclonal antibody was used alone. This confirmed that monocytes differentiated into M1 macrophages more when treated in combination with anti-PD-1 antibody and/or anti-CD47 antibody than when anti-CD300c monoclonal antibody was used alone.

Experimental Example 7.3. Confirmation of Reduction of M2 Macrophage Markers In order to confirm whether the induction of differentiation of monocytes into M2 macrophages is reduced when anti-CD300c monoclonal antibody is administered in combination with immunological anticancer drugs such as Imfinzi, Opdivo, Keytruda, anti-CTLA-4, or αCD47, ^1.5x104 cells/well of THP-1 were dispensed into a 96-well plate, pretreated with 320nM PMA for 6 hours, and then treated with 10μg/mL of anti-CD300c monoclonal antibody and immunological anticancer drugs, either alone or in combination, together with 20ng/mL of IL-4 (Interleukin-4) and IL-13 (Interleukin-13), and reacted for 48 hours. Thereafter, the production amounts of IL-10 and IL-12, which are differentiation markers of M2 macrophages, were measured using an ELISA kit (R&D Systems).

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

Experimental Example 7.4. Confirmation of Increase in M1 Macrophage Differentiation Ability In order to confirm that the differentiation ability of monocytes into M1 macrophages increases when CL7, an anti-CD300c monoclonal antibody, is treated in combination with an immunological anticancer drug such as anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, or anti-CD47 antibody, the signal transduction of MAPK (mitogen-activated protein kinase), IkB, and NF-kB, which are representative signals of M1 macrophage differentiation, was confirmed. Specifically, 8.8 x ¹⁰⁵ cells/well of THP-1 were dispensed into a 6-well plate and treated with 10 μg/mL of anti-CD300c monoclonal antibody, 10 μg/mL of Imfinzi, and/or 10 μg/mL of Keytruda. As a control group, the same amount of phosphate buffer solution (PBS) was used. After 48 hours of culture, phosphorylated SAPK/JNK, phosphorylated ERK, and phosphorylated p38 were confirmed in the case of MAPK signaling, phosphorylated NF-kB in the case of NF-kB signaling, and phosphorylated IkB in the case of IkB signaling by Western blotting. The results are shown in Figures 53 to 55.

Figures 53, 54 and 55 show the results of confirming the signal transduction of MAPK, NF-Kb and IkB, respectively. It was confirmed that the amount of phosphorylated MAPK, IkB and NF-kB increased when anti-CD300c was treated in combination with immune anticancer drugs 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. From this, it was confirmed that the cell signal transduction to differentiate into M1 macrophages increased when anti-CD300c monoclonal antibody was treated in combination with immune anticancer drugs 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.

Experimental Example 8. Confirmation of (synergistic) increase in the cancer cell growth inhibitory effect by combined use (In vitro)
Experimental Example 8.1. Confirmation of apoptosis signaling We confirmed whether apoptosis signaling was increased when anti-CD300c monoclonal antibody CL7 was treated in combination with immunological anticancer drugs such as anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, and anti-CD47 antibody. Specifically, ^8x105 cells/well of A549 were dispensed into a 6-well plate and treated with 10μg/mL of anti-CD300c monoclonal antibody, 10μg/mL of Imfinzi, Keytruda, Opdivo, and anti-CD47 antibody, either alone or in combination. After 48 hours of culture, apoptosis signaling or cell cycle signaling was confirmed by Western blotting. The apoptosis signaling markers identified were cleaved caspase-9, caspase-3, caspase-2, and caspase-8, and the cell cycle signaling markers identified were cyclin D1, CDK2, p27kip1, CDK6, cyclin D3, P21 Waf1, Cip1, etc.

As shown in Figure 56, the apoptosis signal was increased when anti-CD300c monoclonal antibody was combined with Imfinzi, an anti-PD-1 antibody, compared to when it was used alone, and the amount of cleaved-caspase 9 and p21 increased and cyclin D1 decreased when anti-CD300c monoclonal antibody was combined with immune anticancer drugs such as anti-PD1, anti-PD-L1, anti-CTLA-4, and anti-CD47. This confirmed that apoptosis of cancer cells was more effectively induced when anti-CD300c monoclonal antibody was combined with immune anticancer drugs such as anti-PD-1, anti-PD-L1, anti-CTLA-4, and anti-CD47 antibodies, compared to when it was used alone.

Experimental Example 8.2. Confirmation of the growth inhibitory effect on cancer cell lines In order to confirm the growth inhibitory effect of the anti-CD300c monoclonal antibody CL7 and the immunological anticancer agent, the growth inhibitory effect on cancer cells was compared using A549 (human lung cancer cell line) and MDA-MB-231 (human breast cancer cell line). Specifically, ^2x104 cells (A549) or ^3x104 cells (MDA-MB-231) were dispensed into a 96-well plate without fetal bovine serum (FBS), and ^6x103 cells (A549) or ^1x104 cells (MDA-MB-231) were dispensed into a 96-well plate with 0.1% fetal bovine serum. Then, the cells were treated with 10μg/mL of anti-CD300c monoclonal antibody and Imfinzi alone or in combination, and cultured for 5 days. As a control, the same amount of phosphate buffer solution (PBS) was added. Then, CCK-8 (DOJINDO) was added and the absorbance was measured at OD 450 nm. The results are shown in Figure 57 (A549) and Figure 58 (MDA-MB-231).

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

When MDA-MB-231 cell line was treated, as shown in FIG. 58, 19% higher cancer cell growth inhibition effect was observed when anti-CD300c monoclonal antibody was treated alone with 0.1% FBS compared to the control group, 45% higher inhibition effect was observed when anti-CD300c monoclonal antibody and anti-CD47 antibody were treated in combination, and 51% higher inhibition effect was observed when anti-CD300c monoclonal antibody, anti-CD47 antibody, and Imfinzi were treated together. Under 0.1% FBS conditions, 19% higher cancer cell growth inhibition effect was observed when anti-CD300c monoclonal antibody was treated with 0.1% FBS, 22% higher inhibition effect was observed when anti-CD300c monoclonal antibody and anti-CD47 antibody were treated in combination, and 32% higher inhibition effect was observed when anti-CD300c monoclonal antibody, anti-CD47 antibody, and Imfinzi were treated together.

These results confirmed that the growth of cancer cells was further suppressed when anti-CD300c monoclonal antibody was used in combination with immunological anticancer drugs such as anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, and anti-CD47 antibody, compared to when it was used alone.

Experimental Example 9. Confirmation of (synergistic) increase in in vivo anticancer effect by combined use (colon cancer mouse model)
Experimental Example 9.1. Confirmation of in vivo cancer growth inhibitory effect In order to confirm the anti-cancer effect of anti-CD300c monoclonal antibody CL7 under in vivo conditions, ^2x105 colon cancer cell lines (CT26) were subcutaneously implanted into 8-week-old BALB/c mice to prepare an allograft mouse tumor model. All animal breeding and experiments were carried out in an SPF facility. On the 12th day (D12) after implantation of the colon cancer cell lines, anti-CD300c monoclonal antibody and anti-PD-1 antibody purchased from BioXcell were administered alone or in combination to mice with tumors measuring 50-100 ^mm3 , and the same amount of phosphate buffered saline (PBS) was injected as a control. A schematic experimental method is shown in FIG. 59. Specifically, mice were intraperitoneally injected with each antibody, either alone or in combination, twice a week for 2 weeks, a total of 4 times (D12, D15, D19, and D22) (CL7: 10 mg/kg; anti-PD-1 antibody: 10 mg/kg). Tumor volumes were measured for 25 days. The results are shown in FIG.

As can be seen from Figure 60, the experimental group in which anti-CD300c monoclonal antibody was administered alone also had suppressed cancer growth compared to the control group, but it was confirmed that cancer growth was more effectively suppressed when anti-CD300c monoclonal antibody was administered in combination with an immunological anticancer drug such as anti-PD-1 antibody than when it was administered alone.

Experimental Example 9.2. Confirmation of Increase in Tumor Infiltrating Lymphocytes in In Vivo Tumor Microenvironment To confirm the effect of anti-CD300c monoclonal antibody on tumor infiltrating lymphocytes (TIL) in the tumor microenvironment (TME), mice were euthanized on the 25th day after the experiment in the same manner as in Experimental Example 9.1, and 1% PFA (para-formaldehyde) was intravascularly injected into the mice for perfusion, and cancer tissue was obtained. The obtained cancer tissue was fixed using 1% PFA and dehydrated using 10%, 20%, and 30% sucrose solutions in sequence. The dehydrated cancer tissue was frozen in OCT compound (Optimal cutting temperature compound) and then cut into 50 μm-thick sections using a cryotome. The tissue was incubated with a mixture of 20 mg/ml collagenase D and 2 mg/ml DNase I at 37°C for 1 hour, filtered through a 70 um cell strainer, red blood cells were lysed, and the cells were refiltered through a nylon mesh to dissolve the cells into single cells. The single cell suspension was reacted with CD16/32 (obtained from Invitrogen) antibody for 1 hour to suppress non-specific reactions, confirming cell viability, and staining tumor-infiltrating lymphocyte markers CD8+ T cells and CD31+ cancer vascular cells.

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 anti-CD300c exerts an anti-cancer effect by increasing tumor-infiltrating lymphocytes in the tumor microenvironment when 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, more than when administered alone.

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

As a result, as shown in FIG. 61, M1 macrophages were partially increased in the experimental group treated with anti-PD-1 antibody compared to the control group, but M1 macrophages were significantly increased in the experimental group treated with anti-CD300c monoclonal antibody, and M2 macrophages were hardly observed. In addition, it was confirmed that M1 macrophages were increased more in the experimental group administered with a combination of anti-CD300c monoclonal antibody and anti-PD-1 antibody. From the above results, it was confirmed that the differentiation into M1 macrophages is more effectively promoted when anti-CD300c monoclonal antibody is administered in combination with an immunological anticancer agent such as anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, or anti-CD47 antibody than when it is administered alone.

Experimental Example 9.4. Confirmation of the in vivo CD8+ T cell immunity promoting effect In order to confirm whether the anti-CD300c monoclonal antibody CL7 promotes CD8+ T cell immunity in a mouse tumor model, mice were euthanized on the 25th day of the experiment in the same manner as in Experimental Example 9.1, and 1% PFA was intravascularly injected into the mice for perfusion, and cancer tissue was obtained. The obtained cancer tissue was fixed using 1% PFA and dehydrated in 10%, 20%, and 30% sucrose solutions in sequence. The dehydrated cancer tissue was frozen in OCT compound, and then cut into 50 μm-thick sections using a frozen tissue sectioner. Then, CD8+ and iNOS were stained.

As shown in FIG. 62, the number of CD8+ T cells was increased slightly in the experimental group treated with anti-PD-1 antibody compared to the control group, but the number of CD8+ T cells was significantly increased in the experimental group treated with anti-CD300c monoclonal antibody. In addition, the number of CD8+ T cells was increased more in the experimental group treated with anti-CD300c monoclonal antibody and anti-PD-1 antibody than in the group treated with anti-PD-1 alone. From the above results, it was confirmed that anti-CD300c monoclonal antibody can more effectively increase the number of CD8+ T cells when used in combination with existing immunotherapy anticancer drugs.

Experimental Example 9.5. Confirmation of the effect of increasing in vivo immune cell activity In order to confirm whether the activity of immune cells increases when anti-CD300c monoclonal antibody is administered in combination with an immune anticancer drug, spleens were obtained from mice that had been administered 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, or anti-CD47 antibodies in the same manner as in Experimental Example 9.1. The obtained spleen was FACS stained with various markers that indicate the activity of T cells and NKT cells as described in Experimental Example 9.2, and confirmed through MFI.

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

Experimental Example 9.6. Confirmation of in vivo Treg suppressive effect In order to confirm the change in the appearance of Treg cells that cause immune suppressive reactions 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, anti-CD47 antibodies were administered in combination in a solid cancer model, T cells were extracted from spleen tissue prepared in the same manner as in Experimental Example 9.2, and the number of Tregs expressing FOXP3 among CD3+ T cells was confirmed by FACS staining.

When the ratio of Tregs in CD3+ cells was examined, it was confirmed that the ratio of Tregs was significantly reduced when each immunotherapy anticancer 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.

Experimental Example 10. Confirmation of (synergistic) increase in in vivo anticancer effect by combined use (melanoma mouse model)
Experimental Example 10.1. Confirmation of in vivo cancer growth inhibitory effect In order to confirm whether the anti-CD300c monoclonal antibody CL7 is effective in other carcinomas besides the CT26 colon cancer mouse model, an additional experiment was carried out in a melanoma mouse model. An allograft mouse tumor model was prepared by subcutaneously injecting ^7x105 B16F10 melanoma cells into 8-week-old male C57BL/ ⁶ mice. All animal breeding and experiments were carried out in an SPF facility. Eight days after the melanoma cell line was inject- ed, 25 mg/kg CL7, 10 mg/kg α-PD-1, and 4 mg/kg α-CTLA4 were intraperitoneally injected into mice with tumors measuring 50-100 mm3. A schematic experimental method is shown in FIG. 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), and as a control group, phosphate buffered saline (PBS) was injected in the same amount as the anti-CD300c monoclonal antibody. The size of the tumor was measured for 20 days.

As a result, as shown in FIG. 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.

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

Tumor tissues were collected from 6 mice per group. After removal, the tumor tissues were incubated in a mixture of collagenase D (20 mg/ml) and DNase I (2 mg/ml) at 37°C for 1 hour. They were then filtered through a 70 μm cell strainer, red blood cells were lysed, and then refiltered through a nylon mesh. The single cell suspension was then blocked with CD16/32 (obtained from Invitrogen) antibody, 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.

As a result, as shown in FIG. 65, it was confirmed that the number of CD8+ T cells was increased in the B16F10 melanoma model by combined administration of anti-CD300c monoclonal antibody and anticancer immunotherapy (groups D and T), similar to the CT26 carcinoma model (Experimental Example 6.2). Here, group D shows the combined use of CL7 and anti-PD-1 antibody, and group T shows the combined use of CL7, anti-PD-1 antibody, and anti-CTLA-4 antibody.

Experimental Example 10.3. Confirmation of Increase in Cytotoxic T Cells Compared to Regulatory T Cells To confirm the effect of the combination of anti-CD300c monoclonal antibody CL7 with anti-PD-1 antibody and/or anti-CTLA-4 antibody on regulatory T cells in a B16F10 melanoma model under in vivo conditions, a mouse tumor model was prepared as in Experimental Example 10.1 and each test substance was injected at the same concentration. The experimental groups were (i) a CL7 administration group, (ii) an anti-PD-1 administration group, (iii) a CL7 and anti-PD-1 administration group (group D), and (iv) a CL7, anti-PD-1 antibody, and anti-CTLA4 antibody administration group (group T).

Tumor tissues were collected from six mice per group. After removal, the tumor tissues were incubated in a mixture of collagenase D (20 mg/ml) and DNase I (2 mg/ml) at 37°C for 1 hour. They were then filtered through a 70 μm cell strainer, red blood cells were lysed, and then refiltered through a nylon mesh. The single cell suspension was then blocked with CD16/32 (obtained from Invitrogen) antibody, and the cells were stained with a cell viability stain, antibodies against 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.

As a result, as shown in FIG. 66, it was confirmed that, similar to the CT26 carcinoma model (Experimental Example 6.5), the combined administration of anti-CD300c monoclonal antibody and an immunological anticancer agent (groups D and T) also increased CD8+ T cells compared to regulatory T cells in the B16F10 melanoma model. Here, group D shows the combined use of CL7 and anti-PD-1 antibody, and group T shows the combined use of CL7, anti-PD-1 antibody, and anti-CTLA-4 antibody.

Experimental Example 10.4. Confirmation of Increase in Tumor-Associated Macrophages (TAM) To confirm the 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 under in vivo conditions, a mouse tumor model was prepared as in Experimental Example 10.1 and each test substance was injected at the same concentration.

Tumor tissues were collected from six mice per group. After removal, the tumor tissues were incubated in a mixture of collagenase D (20 mg/ml) and DNase I (2 mg/ml) at 37°C for 1 hour. They were then filtered through a 70 μm cell strainer, red blood cells were lysed, and then filtered again through a nylon mesh. The single cell suspension was then blocked with CD16/32 (obtained from Invitrogen) antibody, 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.

As a result, as shown in FIG. 67, it was confirmed that the expression level of M1 tumor-associated macrophages was increased in the B16F10 melanoma model by combined administration of anti-CD300c monoclonal antibody and anticancer immunotherapy (groups D and T), similar to the CT26 carcinoma model (Experimental Example 6.1). Here, group D shows the combined use of CL7 and anti-PD-1 antibody, and group T shows the combined use of CL7, anti-PD-1 antibody, and anti-CTLA-4 antibody.

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

Experimental Example 11. Confirmation of complete remission phenomenon in a mouse model of colon cancer by combined administration In order to confirm the anti-cancer effect of combined administration of anti-CD300c monoclonal antibody CL7 and anti-PD-1 antibody and/or anti-CTLA- ⁴ antibody under in vivo conditions, 2x105 colon cancer cell lines (CT26) were subcutaneously transplanted into 8-week-old BALB/c mice to prepare an allograft mouse tumor model. All animal breeding and experiments were carried out in an SPF facility. On the 11th day (D11) after transplantation of the colon cancer cell lines, mice with tumors measuring ^50-100 mm3 were administered anti-CD300c monoclonal antibody, anti-PD-1 antibody, and anti-CTLA-4 antibody purchased from BioXcell, either alone or in combination, and the control group was injected with the same amount of phosphate buffered saline (PBS) as CL7. Specifically, on D11, D14, and D18, 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 volumes were measured.

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

As shown in FIG. 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 provides an excellent anti-cancer effect.

Experimental Example 12. Confirmation of the effect of combined administration on improving long-term survival rate The long-term survival rate of the mice tested in Experimental Example 11 was confirmed. As a result, as shown in Figure 69, it was confirmed that the long-term survival rate was improved when the anti-CD300c monoclonal antibody was combined with an immunological anticancer agent such as an anti-PD-1 antibody or an anti-CTLA-4 antibody, compared to when the anti-CD300c monoclonal antibody was used alone.

Experimental Example 13. Confirmation of the effect of combined administration on preventing cancer recurrence In order to confirm the effect of combined administration of anti-CD300c monoclonal antibody CL7 and an immunosuppressant on preventing cancer recurrence in vivo, ^2x105 colon cancer cell lines (CT26) were subcutaneously implanted into 8-week-old BALB/c mice to prepare homograft mouse tumor models, and the experiment was carried out as described in Experimental Example 11 to obtain mice in complete remission. ^2x105 colon cancer cell lines (CT26) were re-implanted (Re-challenge) into the obtained mice, and the mice were observed for 30 days.

As a result, as shown in Figure 70, it was confirmed that the group that achieved complete remission through the combined administration of anti-CD300c monoclonal antibody and immune anticancer drug did not experience cancer recurrence or metastasis. This suggests that individuals who achieved complete remission through combined administration of anti-CD300c monoclonal antibody and anti-PD-1 antibody (CL7 + αPD-1; Combi) or triple combination administration with the addition of anti-CTLA-4 antibody (CL7 + αPD-1 + αCTLA-4; Triple) would exhibit systemic defensive immune responses through sustained immune memory, thereby suppressing cancer recurrence and metastasis.

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

As a result, as shown in FIG. 71, it was confirmed that effector memory T cells were significantly increased when anti-CD300c monoclonal antibody and an immunological anticancer drug were administered in combination. This indicates that, as predicted in Experimental Example 13, mice that achieved complete remission had immune memory through the increased effector memory T cells when CL7 and anti-PD-1 antibody were administered in combination (Combi) or when anti-CTLA-4 antibody was also administered in triple combination (Triple), and thus suppressed the growth of additional cancer cells even if they were generated.

Example 4. Concomitant administration of anti-CD300c monoclonal antibodies (CL10, SL18) and immunological anticancer agents The anti-CD300c monoclonal antibodies (CL10, SL18) prepared in Example 1 were administered in combination with other immunological anticancer agents, such as Imfinzi, an anti-PD-L1 antibody, and Keytruda, an anti-PD-1 antibody, and the results were observed.

The sources of these immunotherapy drugs are as follows: Imfinzi (AstraZeneca) and Keytruda (Merck Sharp & Dohme).

Experimental Example 15. Confirmation of Increase in M1 Macrophage Differentiation Ability To confirm whether anti-CD300c monoclonal antibodies CL10 or SL18 can promote differentiation of macrophages into M1 macrophages, ^1x104 cells/well of THP-1 cells were dispensed into a 96-well plate and treated with 10μg/mL of CL10 or SL18. To confirm the effect of combined treatment of CL10 or SL18 with an immunological anticancer drug, the anti-CD300c monoclonal antibody was treated with 10μg/mL of Imfinzi and/or Keytruda. After incubation for 48 hours in a _CO2 incubator, the amount of TNF-α produced, which is a differentiation marker for M1 macrophages, was confirmed 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).

As shown in Figures 72a and 72b, it was confirmed that the expression level of TNF-α was increased when THP-1 cells were treated with CL10 or SL18 in combination with Imfinzi or Keytruda compared to when they were treated alone. In particular, it was confirmed that the expression level of TNF-α was highest when CL10 or SL18 was administered in combination with the two antibodies Imfinzi and Keytruda at the same time.

Experimental Example 16. Confirmation of Cancer Cell Growth Inhibitory Effect To confirm the cancer cell growth inhibitory effect of the combined administration of anti-CD300c monoclonal antibodies CL10 or SL18 and immunological anticancer drugs, the cell growth inhibitory effect was compared using A549 (human lung cancer cell line) cells. Specifically, ^2x104 cells were dispensed into a 96-well plate without FBS, and ^6x103 cells were dispensed into a 96-well plate with 0.1% FBS. Then, CL10 or SL18 was treated alone or in combination with Imfinzi and/or Keytruda at a concentration of 10μg/mL, respectively, and cultured for 5 days. Then, 30ul of CCK-8 (DOJINDO) was treated per well, and the absorbance was measured at O.D 450nm every hour while incubating in a _CO2 incubator for 4 hours. The results are shown in Figure 73a (combined with CL10) and Figure 73b (combined with SL18).

As shown in Figures 73a and 73b, when A549 cells were treated with 0.1% FBS in combination with Imfinzi or Keytruda, the cancer cell proliferation inhibitory effect was further increased compared to when CL10 or SL18 was treated alone, and it was confirmed that the cancer cell proliferation inhibitory effect was the best when CL10 or SL18 was administered in combination with the two antibodies Imfinzi and Keytruda at the same time.

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 exhibit efficacy through the same mechanism of action as CL7, and, like CL7, their efficacy was increased by co-administration with an immunosuppressant.

Statistical Processing The results obtained through the experiment were analyzed for comparison between the experimental groups using one-way analysis of variance followed by Bonferroni post-hoc test. When the p-value was 0.05 or less, the difference between the groups was significant.

Claims (42)

(i) a CDR1 comprising 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;
CDR2 comprising 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 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 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;
CDR2 comprising 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 An anti-CD300c monoclonal antibody or antigen-binding fragment thereof, comprising a light chain variable region comprising a CDR3 comprising 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. The heavy chain variable region comprises:
(i) a CDR1 comprising 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 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 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 comprises:
(ii) a CDR1 comprising 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;
2. The anti-CD300c monoclonal antibody or antigen-binding fragment thereof according to claim 1, comprising: a CDR2 comprising 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 a CDR3 comprising 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. The heavy chain variable region comprises: (i) a CDR1 comprising 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;
CDR2 comprising 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 CDR3 comprising 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 comprises (ii) a CDR1 comprising 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;
The anti-CD300c monoclonal antibody or antigen-binding fragment thereof according to claim 1, comprising: a CDR2 comprising 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 a CDR3 comprising 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. 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 a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 46, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 47, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 48. The heavy chain variable region comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 79, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 80, and a CDR3 comprising the amino acid sequence represented by 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 a CDR1 comprising the amino acid sequence represented by SEQ ID NO: 82, a CDR2 comprising the amino acid sequence represented by SEQ ID NO: 83, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO: 84. 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 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. the heavy chain variable region comprises a CDR1 comprising the amino acid sequence represented by SEQ ID NO:211, a CDR2 comprising the amino acid sequence represented by SEQ ID NO:212, and a CDR3 comprising the amino acid sequence represented by 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 a CDR1 comprising the amino acid sequence represented by SEQ ID NO:214, a CDR2 comprising the amino acid sequence represented by SEQ ID NO:215, and a CDR3 comprising the amino acid sequence represented by SEQ ID NO:216. 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;
The anti-CD300c monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein 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. 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 of 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. 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. The anti-CD300c monoclonal antibody or antigen-binding fragment thereof according to claim 10, wherein the anti-CD300c monoclonal antibody or antigen-binding fragment thereof is cross-reactive with both human and mouse CD300c antigens. An anti-CD300c monoclonal antibody or an antigen-binding fragment thereof, comprising: a heavy chain variable region comprising CDR1 to CDR3 having amino acid sequences represented by the following formulas (1) to (3), respectively; and a light chain variable region comprising CDR1 to CDR3 having amino acid sequences represented by the following formulas (4) to (6), respectively:
FTFX1X2X3X4MX5WVR (1)
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)
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)
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)
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 not present X9 = R, S, G, or not present X10 = N or not present X11 = Y or not present X12 = N, H, or Q
X1X2X3X4RPSGVX5 (5)
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)
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. A pharmaceutical composition for preventing or treating cancer, comprising as an active ingredient the anti-CD300c monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 12. The pharmaceutical composition according to claim 13, wherein the cancer includes 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. The pharmaceutical composition according to claim 13, wherein the cancer is a solid cancer. The pharmaceutical composition according to claim 13, wherein the cancer comprises one or more selected from the group consisting of colorectal cancer, lung cancer, melanoma, and breast cancer. The pharmaceutical composition according to claim 13, wherein the pharmaceutical composition suppresses cancer proliferation, survival, metastasis, recurrence, or resistance to an anticancer drug. The pharmaceutical composition according to claim 13, further comprising one or more immunological anti-cancer agents. The pharmaceutical composition according to claim 18, wherein the immunological anticancer 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. The pharmaceutical composition according to claim 18, wherein the immunological anticancer agent comprises one or more selected from the group consisting of anti-PD-1, anti-PD-L1, anti-CTLA-4 and anti-CD47 antibodies. The pharmaceutical composition according to claim 18, wherein the immunological anticancer agent comprises one or more selected from the group consisting of durvalumab, pembrolizumab, nivolumab, αCD47, and ipilimumab. The pharmaceutical composition according to claim 18, wherein the anti-CD300c antibody or its antigen-binding fragment and the immunological anticancer agent are each formulated and administered simultaneously or sequentially separately. A method for preventing or treating cancer, comprising administering an anti-CD300c monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12 to a subject in need of cancer prevention or treatment. 24. The method of claim 23, further comprising administering one or more immunological anti-cancer agents. The method according to claim 23, further comprising a step of confirming the expression level of CD300c protein based on a biological sample or data from the subject prior to administration of the anti-CD300c monoclonal antibody or antigen-binding fragment thereof. The method according to claim 25, 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. The method of claim 23, further comprising determining the expression level of one or more markers selected from the following markers using a biological sample or data from a subject to which the anti-CD300c monoclonal antibody or antigen-binding fragment thereof has been administered:
Bst2, Cd40, Cd70, Cd86, Ccl8, Xcl1, Ccr7, Cd80, Cd206, Msr1, Arg1, Vegfa, Pdgfrb, Col4a1, 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. The method of claim 27, further comprising the step of selecting one or more immunological anticancer agents to be used in combination with the anti-CD300c monoclonal antibody or antigen-binding fragment thereof based on the expression level of the identified marker. The method of claim 28, 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. The method of claim 27, 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. The method according to claim 30, 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 vegfa, pdgfrb, Col4a1, Hif1a, and IL-6 among the markers is statistically significantly decreased compared to a subject not administered the anti-CD300c antibody or its antigen-binding fragment. The method according to claim 30, 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 with the anti-CD300c antibody or its antigen-binding fragment. A composition comprising the anti-CD300c monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 12, and an instruction manual for use in the prevention or treatment of cancer. The kit of claim 33, 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. The kit of claim 33, wherein the instructions include instructions for measuring the expression level of CD300c protein using a biological sample or material obtained from the subject prior to administration of the antibody or antigen-binding fragment thereof. A method for providing information for preventing or treating cancer, comprising the step of measuring the expression level of CD300c protein based on a biological sample or material obtained from a subject in need of cancer prevention or treatment. The method according to claim 36, wherein the information for preventing or treating cancer includes information on one or more of therapeutic responsiveness to a therapeutic agent (e.g., anti-CD300c or an antigen-binding fragment thereof) associated with CD300c protein, selection of a therapeutic agent, selection of a subject to be treated, prognosis of the subject, and survival time of the subject. 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 material obtained from a subject in need of cancer prevention or treatment. An isolated nucleic acid molecule encoding an anti-CD300c monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 12. An expression vector comprising the nucleic acid molecule of claim 39. A host cell comprising the nucleic acid molecule of claim 39. A method for producing an anti-CD300c monoclonal antibody or an antigen-binding fragment thereof, comprising culturing the host cell according to claim 41.