JP7453697B2 - Immune checkpoint inhibitor antibody - Google Patents

Description

The present invention relates to immune checkpoint inhibiting antibodies.

Since the discovery of cancer antigens, various research efforts have been underway toward the development of cancer immunotherapy. In conventional cancer immunotherapy, various adjuvants, cancer antigen proteins and cancer antigen peptides are used to induce anti-tumor immune responses. However, because many cancer antigens are normal self-antigens, their immunogenicity is weak, and in addition, the immune control mechanism that suppresses the immune response to self-antigens (autoimmunity) also suppresses the immune response to cancer antigens. It is becoming clear that there is. Such immune control mechanisms are carried out by molecules called "immune checkpoint molecules." Immune checkpoint molecules negatively regulate antitumor immune responses and autoimmune responses, and examples include CTLA-4 and PD-1 expressed in T cells (Non-Patent Document 1). Immunotherapy methods using inhibitory antibodies against these "immune checkpoint molecules" have been attempted (Non-Patent Document 1). However, although such immunotherapy methods have shown some clinical efficacy, it has been pointed out that many cancer types remain unresponsive, and results are still unsatisfactory. I can't say that. Furthermore, inhibition of the immune checkpoint molecule "CTLA-4/PD-1 system" expressed in T cells causes adverse events such as autoimmune-like pathologies caused by activation of immune responses against not only cancer but also the self. There are concerns that this will happen. In this way, the development of immune checkpoint inhibitors aims to not only overcome cancer unresponsiveness but also reduce immune-related adverse events (irAEs) caused by breakdown of autoimmune tolerance. is also in demand.

Dendritic cells (DCs) are antigen-presenting cells that have dendrites, are negative for lineage markers, and are positive for major histocompatibility complex class II, and have characteristics similar to conventional dendritic cells (cDCs). It consists of a subpopulation that is broadly classified as plasmacytoid dendritic cells (plasmacytoid DCs; pDCs). In inflammatory conditions, dendritic cells act as the most powerful antigen-presenting cells that connect innate immunity and adaptive immunity, activating the immune system through the induction of antigen-specific effector T cells, and in steady states, eliminating antigen-specific clones and nonresponsiveness. They play an important role in maintaining immunological homeostasis as regulatory cells that induce immune tolerance through the generation and expansion of regulatory T (Treg) cells, which have the ability to induce sex and suppress immunity. On the other hand, it has been speculated that the inhibition of dendritic cell function in the cancer environment reduces the cancer immune response, but many aspects of its mechanism of action remain unclear.

The present inventors have succeeded in producing immunosuppressive dendritic cells that have remarkable T cell control functions even in inflammatory conditions in humans and mice under specific culture conditions (Non-patent Documents 2 and 3).

The present inventors also identified "Clec4A4 (DCIR2)", which belongs to the C-type lectin receptor family, and found that Clec4A4 controls dendritic cell function through intracellular signal transduction and attenuates inflammation and T cell immunity. It was shown that (Non-Patent Document 4). It has also been found that in Clec4A4-deficient mice, the onset of autoimmune diseases is accelerated due to excessive expansion and activation of autoreactive T cells, and autoimmune pathology is exacerbated (Non-Patent Document 4).

Topalian S.L., et al., Cancer Cell. (2015) 27:450-461 Sato K., et al., Immunity. (2003) 18:367-379 Sato K., et al., Blood. (2003) 101:3581-3589 Uto T., et al., Nat. Commun., (2016) 7:11273

An object of the present invention is to provide an immune checkpoint inhibiting antibody based on a new mechanism that is useful for activating anti-tumor immune responses.

As a result of intensive studies to solve the above problems, the present inventors discovered that a specific antibody that binds to the extracellular region of the human CLEC4A protein has a high antitumor effect due to its immune checkpoint inhibiting action. , we have completed the present invention.

That is, the present invention includes the following.
[1] An antibody or antigen-binding fragment thereof that specifically binds to the extracellular region of human CLEC4A protein and has an immune checkpoint inhibiting effect.
[2] The antibody or antigen-binding fragment thereof according to [1] above, which specifically binds to a region consisting of the amino acid sequence shown by SEQ ID NO: 21 in the extracellular region of human CLEC4A protein.
[3] The antibody or antigen-binding fragment thereof according to [1] or [2] above, which specifically binds to a peptide fragment containing the amino acid sequence WVDQTPYNESSTFW (SEQ ID NO: 15).
[4] Any of the above [1] to [3] that can bind to a peptide fragment consisting of a 15 amino acid long amino acid sequence including WVDQTPYNESSTFW (SEQ ID NO: 15) on the amino acid sequence shown by SEQ ID NO: 2. The antibody or antigen-binding fragment thereof as described.
[5] Can bind to a peptide fragment consisting of a 15 amino acid long amino acid sequence including QNLQEESAYF (SEQ ID NO: 16) on the amino acid sequence shown in SEQ ID NO: 2 and a peptide fragment consisting of an amino acid sequence FSSNCYFISTESASW (SEQ ID NO: 17). , the antibody or antigen-binding fragment thereof according to [1] or [2] above, which binds to discontinuous epitopes of human CLEC4A protein.
[6] A peptide fragment consisting of a 15 amino acid long amino acid sequence containing QNLQEESAYF (SEQ ID NO: 16) on the amino acid sequence shown by SEQ ID NO: 2 and SSNCYFISTESASW (SEQ ID NO: 18) on the amino acid sequence shown by SEQ ID NO: 2 The antibody or antigen-binding fragment thereof according to [1] or [2] above, which is capable of binding to a peptide fragment consisting of an amino acid sequence of 15 amino acids in length and binds to discontinuous epitopes of human CLEC4A protein.
[7] The antibody or antigen-binding fragment thereof according to [1] above, which specifically binds to a peptide fragment containing the amino acid sequence KKNMPVEETAWSCC (SEQ ID NO: 19).
[8] The antibody according to [1] or [7] above, which is capable of binding to a peptide fragment consisting of a 15 amino acid long amino acid sequence containing KKNMPVEETAWSCC (SEQ ID NO: 19) on the amino acid sequence shown by SEQ ID NO: 2. or an antigen-binding fragment thereof.
[9] An antitumor drug comprising the antibody or antigen-binding fragment thereof according to any one of [1] to [8] above.
[10] The antitumor drug according to [9] above, for use in combination with an inhibitor against immune checkpoint molecules expressed in T cells.
[11] The antitumor drug according to [9] above, further comprising an inhibitor against immune checkpoint molecules expressed in T cells.
[12] The antitumor drug according to [10] or [11] above, wherein the immune checkpoint molecule expressed on T cells is PD-1.
[13] The antitumor drug according to any one of [10] to [12] above, wherein the inhibitor against immune checkpoint molecules expressed in T cells is an anti-PD-1 antibody.
[14] The antitumor drug according to any one of [9] to [13] above, which is used to suppress the progression of cancer.

This specification includes the disclosure content of Japanese Patent Application No. 2019-209977, which is the basis of the priority of this application.

According to the present invention, it is possible to provide antibodies that can activate anti-tumor immune responses through immune checkpoint inhibition and bring about anti-tumor effects.

Figure 1 shows OVA-specific T cells (CD44 ^high OVA-MHC class I pentamer-binding CD8 The results of flow cytometry analysis of the induction of T cells) ^are shown. A is a dot plot showing CD44 on the Y axis and OVA-MHC class I pentamer on the X axis, B shows the percentage (%) of CD44 ^high OVA-MHC class I pentamer-binding CD8 ⁺ T cells among CD8 ⁺ T cells. . *P<0.01 (comparison with WT mice or comparison between non-tumor-bearing and tumor-bearing groups in WT and Clec4A4-deficient mice). Figure 2 shows OVA-specific T cells (OVA-specific IFN-γ-producing CD8 ⁺ T The results of analysis using flow cytometry method regarding the induction of cells (cells) are shown. A is a dot plot with CD8α on the Y axis and IFN-γ on the X axis, and B shows the percentage (%) of OVA-specific IFN-γ-producing CD8 ^{+ T cells among CD8 + T} cells. *P<0.01 (comparison with WT mice or comparison between non-tumor-bearing and tumor-bearing groups in WT and Clec4A4-deficient mice). FIG. 3 shows the evaluation results of cancer progression in tumor-bearing WT mice and Clec4A4-deficient mice that were vaccinated or not vaccinated. A shows a photograph of a tumor-bearing mouse and tumor volume (mm ³ ) 18 days after cancer cell transplantation. B shows temporal changes in tumor volume values 23 days after cancer cell transplantation. C shows the tumor volume 23 days after cancer cell transplantation. *P<0.01 (comparison with vaccinated or non-vaccinated groups of WT mice or comparison between non-tumor-bearing and tumor-bearing groups in WT and Clec4A4-deficient mice). Figure 4 shows the regulatory function of Clec4A4 on immune cell dynamics. Immune cell dynamics in the spleen (A) and cancer tissue (B) of non-tumor-bearing or cancer-bearing (B16-OVA) WT mice and Clec4A4-deficient mice (Clec4a4 ^- / ^- ) 20 days after cancer cell transplantation. Evaluated later. The proportion of each immune cell in spleen cells was analyzed by flow cytometry (A). The proportion of each immune cell in tumor-infiltrating leukocytes was analyzed by flow cytometry (B). The expression of each cell surface molecule is shown as a dot plot. In A and B, the leftmost column is CD11c (X axis) and SiglecH (Y axis), the second column from the left is B220 (X axis) and Gr-1 (Y axis), and the center column is F4/80 (X axis). The second column from the right shows the expression of NK1.1 (X axis) and CD3 (Y axis), the rightmost column shows the expression of CD8 (X axis) and CD4 (Y axis). There is. Figure 5 shows the regulatory function of Clec4A4 on lymphoid tissue T cell activation. T cell dynamics in the spleen of non-tumor-bearing or tumor-bearing (B16-OVA) WT mice and Clec4A4-deficient mice (Clec4a4 ^- / ^- ) was evaluated 20 days after cancer cell transplantation. The expression of each cell surface molecule in splenic CD4 ⁺ T cells (A) and splenic CD8 ⁺ T cells (B) was analyzed by flow cytometry. The expression of each cell surface molecule is shown as a histogram. In A and B, the left column shows the expression of CD44, and the right column shows the expression of CD62L. Figure 6 shows the regulatory function of Clec4A4 on tumor-infiltrating T cell activation. T cell dynamics in cancer tissues of tumor-bearing (B16-OVA) WT mice and Clec4A4-deficient mice (Clec4a4 ^- / ^- ) was evaluated 20 days after cancer cell transplantation. The expression of each cell surface molecule of tumor-infiltrating CD4 ⁺ T cells (A) and tumor-infiltrating CD8 ⁺ T cells (B) was analyzed by flow cytometry. The expression of each cell surface molecule is shown as a histogram. In A and B, the left column shows the expression of CD44, and the right column shows the expression of CD62L. Figure 7 shows the regulatory function of Clec4A4 on splenic dendritic cell activation. Dendritic cell dynamics in the spleen of non-tumor-bearing or tumor-bearing (B16-OVA) WT mice and Clec4A4-deficient mice (Clec4a4 ⁻ / ⁻ ) was evaluated 20 days after cancer cell transplantation. The expression of each cell surface molecule in splenic dendritic cells was analyzed by flow cytometry. The expression of each cell surface molecule is shown as a histogram. In A, the leftmost column shows MHC-I, the second column from the left shows MHC-II, the center column shows CD40, the second column from the right shows CD80, and the rightmost column shows expression of CD86. In B, the leftmost column shows expression of CD11c, the second column from the left shows Clec4A4, the center column shows B7-H1, the second column from the right shows B7-H2, and the rightmost column shows expression of B7-DC. Figure 8 shows the regulatory function of Clec4A4 on tumor-infiltrating dendritic cell activation. Dendritic cell dynamics in cancer tissues of tumor-bearing (B16-OVA) WT mice and Clec4A4-deficient mice (Clec4a4 ^- / ^- ) were evaluated 20 days after cancer cell transplantation. The expression of each cell surface molecule in tumor-infiltrating dendritic cells was analyzed by flow cytometry. The expression of each cell surface molecule is shown as a histogram. In A, the leftmost column shows MHC-I, the second column from the left shows MHC-II, the center column shows CD40, the second column from the right shows CD80, and the rightmost column shows expression of CD86. In B, the leftmost column shows the expression of CD11c, the second column from the left shows Clec4A4, the center column shows B7-H1, the second column from the right shows B7-H2, and the rightmost column shows the expression of B7-DC. FIG. 9 is a diagram showing the expression state of CLEC4A in immune cells derived from the peripheral blood of a healthy person. A shows a histogram of the expression of the cell surface molecule CLEC4A in CD3 ⁺ T cells, CD19 ⁺ B cells, CD11c ⁺ dendritic cells, CD14 ⁺ monocytes, and CD56 ⁺ NK cells in the peripheral blood mononuclear cell fraction. B shows a histogram of the expression of cell surface molecules CD141, CD1c, CD303, CD11c, HLA-DR, and CLEC4A in the peripheral blood mononuclear cell fraction. C is cell surface molecules CD11c, CD40, CD80, CD86, HLA-DR, and CLEC4A in peripheral blood monocytes, peripheral blood monocyte-derived dendritic cells, and peripheral blood monocyte-derived immunosuppressive dendritic cells (DCreg). The expression of is shown as a histogram. Figure 10 is a dot plot showing the expression of GFP and CLEC4A in transgenic cell lines. A is a mouse dendritic cell line DC2.4 (DC2.4 in the figure), a control retrovirus-infected cell line (mock-GFP in the figure), and a CLEC4A-expressing cell line (CLEC4A-GFP in the figure). Expression of GFP (X axis) and CLEC4A (Y axis) is shown. B is a mouse dendritic cell line DC2.4, a cell line expressing a glycosylation site substitution mutant (in the figure, CLEC4A _N185Q -GFP), and a cell line expressing a CRD-EPS deletion mutant (in the figure, CLEC4A _ΔE195-S197 - GFP), a cell line expressing a mutant lacking the extracellular region (in the figure, CLEC4A _ΔF69-L237 -GFP) and a cell line expressing a mutant lacking the ITIM (Immunoreceptor tyrosine-based inhibition motif) sequence (in the figure, CLEC4A _ΔI5-V10 -GFP) ) shows the expression of GFP and CLEC4A. Figure 11 shows cytokine production ability in transgenic cell lines. A shows the amount of IL-6 produced in the culture supernatant in LPS-stimulated or unstimulated CLEC4A or mutant transgenic cell lines. B shows the amount of TNF-α produced in the culture supernatant in LPS-stimulated or unstimulated CLEC4A or mutant transgenic cell lines. C shows the amount of IL-6 produced in the culture supernatant in CLEC4A or mutant transgenic cell lines stimulated or unstimulated with CpG-B ODN 1668. D shows the amount of TNF-α produced in the culture supernatant of CLEC4A or mutant transgenic cell lines stimulated or unstimulated with CpG-B ODN 1668. *P<0.01 (comparison with control retrovirus-infected cell line or comparison between CLEC4A-expressing cell line and glycosylation site substitution mutant-expressing cell line). FIG. 12 shows the positions of the antibody binding sequences (lightly underlined) constituting the epitopes of CLEC4A function-inhibiting antibodies #1 to #4 on the primary sequence of CLEC4A protein. The EPS motif within the carbohydrate recognition domain (CRD) is boldly underlined. The N-type glycosylation site (N185) is indicated by an arrow. Figure 13 shows CLEC4A expression in pancreatic mononuclear cells of CLEC4A-expressing transgenic mice. A shows histograms of CLEC4A expression in CD3 ⁺ T cells, CD19 ⁺ B cells, CD11b ⁺ macrophages, CD11c ⁺ dendritic cells, and NK1.1 ⁺ NK cells in splenic mononuclear cells. Each column of A shows, from left to right, CD3 ⁺ T cells, CD19 ⁺ B cells, CD11b ⁺ macrophages, CD11c ⁺ dendritic cells, and NK1.1 ⁺ NK cells. B shows histograms of CLEC4A expression in splenic dendritic cell subpopulations (CD11c ⁺ CD8α ⁺ cDC1 cells, CD11c ⁺ CD4 ⁺ cDC2 cells, and CD11c ⁺ SiglecH ⁺ pDC cells). Each column in B shows, from left to right, cDC1: CD11c ⁺ CD8α ⁺ cDC1 cells, cDC2: CD11c ⁺ CD4 ⁺ cDC2 cells, and pDC: CD11c ⁺ SiglecH ⁺ pDC cells. FIG. 14 shows the control of CLEC4A function-inhibiting antibodies #1 to #4 on cancer progression in tumor-bearing CLEC4A-expressing transgenic mice. A is a photograph showing the appearance of the tumor 13 days after cancer cell transplantation. B and C show tumor volumes 13 days (B) and 22 days (C) after cancer cell implantation. *P<0.01 (compared to control mouse IgG treated group). FIG. 15 shows changes over time in tumor volume for 22 days after cancer cell transplantation in tumor-bearing CLEC4A-expressing transgenic mice. FIG. 16 shows changes in body weight 22 days after cancer cell transplantation in tumor-bearing mice. FIG. 17 shows the results of analyzing the accumulation of CD4 ⁺ T cells and CD8 ⁺ T cells in cancer tissues derived from cancer-bearing mice by flow cytometry. Expression of each cell surface molecule is shown as a dot plot. Values indicate the percentage of CD4 ⁺ T cells or CD8 ⁺ T cells among tumor-infiltrating leukocytes. FIG. 18 shows the results of tissue staining of each organ of the tumor-bearing mouse 21 days after cancer cell transplantation. FIG. 19 shows the results of cancer progression evaluation. A shows the time course of tumor volume in tumor-bearing mice 21 days after cancer cell transplantation. B shows the tumor volume in tumor-bearing mice 21 days after cancer cell transplantation. C shows a photograph of the tumor-bearing mouse and the excised tumor 21 days after cancer cell transplantation. *P <0.01; compared with untreated group, comparison between each experimental group. FIG. 20 shows the analysis results of cells in cancer tissue. A: Expression of each cell surface molecule is shown as a dot plot. The leftmost column of A is CD11c (X axis) and SiglecH (Y axis), the second column from the left is B220 (X axis) and Gr-1 (Y axis), and the third column from the left is F4/80 ( X-axis) and CD11b (Y-axis), the third column from the right is NK1.1 (X-axis) and CD3 (Y-axis), the second column from the right is CD4 (X-axis) and CD3 (Y-axis), The rightmost column shows the expression of CD8 (X axis) and CD3 (Y axis). B: Shows the expression level of each immunosuppressive molecule in cancer tissue. *P <0.01; compared with untreated group, comparison between each experimental group.

The present invention will be explained in detail below.

The present invention is based on the function of CLEC4A as an immune checkpoint molecule. More specifically, the present invention relates to an antibody that specifically binds to the extracellular region of human CLEC4A protein and has an immune checkpoint inhibitory effect. The present invention also relates to activation of an anti-tumor immune response based on immune checkpoint inhibition using such an antibody and its use in the treatment or prevention of cancer, as well as anti-tumor drugs.

In the present invention, CLEC4A is a protein referred to as C-type (calcium-dependent) lectin domain family 4 Member A. CLEC4A is an immune checkpoint molecule expressed in antigen-presenting cells such as dendritic cells, and has an immunosuppressive function that negatively controls the activation of antigen-presenting cells, thereby negatively regulating cancer immune responses.

Since the antibody having an immune checkpoint inhibitory effect according to the present invention binds to human CLEC4A protein and inhibits the function of CLEC4A as an immune checkpoint molecule, it is also referred to hereinafter as a CLEC4A function-inhibiting antibody. The CLEC4A function-inhibiting antibody according to the present invention binds to the extracellular region of human CLEC4A protein, preferably specifically binds to inhibit its function. A CLEC4A function-inhibiting antibody can bind to and inhibit the function of endogenous CLEC4A protein in a subject to which it is administered. In a preferred embodiment, the CLEC4A function-inhibiting antibody binds to a CLEC4A protein expressed on the cell surface of an antigen-presenting cell, such as a dendritic cell, a B cell (CD19 ⁺ B cell), or a monocyte (CD14 ⁺ monocyte); The function of the CLEC4A protein as an immune checkpoint molecule can be inhibited.

An example of the full-length amino acid sequence of human CLEC4A protein is shown in SEQ ID NO: 2, and an example of the nucleotide sequence encoding it is shown in SEQ ID NO: 1. The human CLEC4A protein may be a human CLEC4A protein consisting of the amino acid sequence shown in SEQ ID NO: 2 or a functional variant thereof. The CLEC4A protein has a sequence identity of 90% or more, 93% or more, 95% or more, or 98% or more, preferably 99% or more, for example 99.5% or more, to the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence. It is preferable that the immune checkpoint function (function as an immune checkpoint molecule in antigen-presenting cells such as dendritic cells) is maintained.

The CLEC4A function-inhibiting antibody according to the present invention may specifically bind to the extracellular region of human CLEC4A protein. In the present invention, the extracellular region of human CLEC4A protein typically refers to the region from position 69 to position 237 of the amino acid sequence shown in SEQ ID NO: 2 (SEQ ID NO: 12), or the amino acid sequence of other CLEC4A proteins. refers to the area that is aligned with that area. In one embodiment, the CLEC4A function-inhibiting antibody may bind (preferably specifically bind) to a carbohydrate-recognition domain (CRD) in the extracellular region of the CLEC4A protein. In the present invention, the sugar chain recognition domain in the extracellular region of the CLEC4A protein is the region from the 107th (Phe) to the 237th (Leu) of the amino acid sequence shown in SEQ ID NO: 2, or other amino acid sequences of the CLEC4A protein. It refers to the area that is aligned with that area in .

In a more preferred embodiment, the CLEC4A function-inhibiting antibody comprises the amino acid sequence (SEQ ID NO: 21) or may bind (preferably specifically bind) to a region comprising or consisting of the same.

The carbohydrate recognition domain (CRD) in the extracellular region of the CLEC4A protein has an EPS motif (Glu-Pro-Ser) at positions 195 to 197 of the amino acid sequence shown by SEQ ID NO: 2 (human CLEC4A protein). The ITIM sequence within the intracellular region of human CLEC4A protein refers to the region located at positions 5 to 10 of the amino acid sequence shown by SEQ ID NO: 2.

In one embodiment, the CLEC4A function-inhibiting antibody (antibody or antigen-binding fragment thereof) according to the present invention consists of a 15 amino acid long amino acid sequence including WVDQTPYNESSTFW (SEQ ID NO: 15) on the amino acid sequence shown by SEQ ID NO: 2. It can (specifically) bind to peptide fragments, namely the peptide fragment consisting of QWVDQTPYNESSTFW (SEQ ID NO: 22) and the peptide fragment consisting of WVDQTPYNESSTFWH (SEQ ID NO: 23). In a preferred embodiment, the CLEC4A function-inhibiting antibody specifically binds to an epitope, preferably a linear (continuous) epitope, comprising the amino acid sequence WVDQTPYNESSTFW (SEQ ID NO: 15) of the human CLEC4A protein. The present invention provides a peptide fragment comprising or consisting of the amino acid sequence WVDQTPYNESSTFW (SEQ ID NO: 15), such as, but not limited to, peptides of 14-20 amino acids in length, 14-15 amino acids in length, or 14 amino acids in length. ) provides a CLEC4A function-inhibiting antibody, which is an antibody that specifically binds to.

In another embodiment, the CLEC4A function-inhibiting antibody (antibody or antigen-binding fragment thereof) according to the present invention consists of a 15 amino acid long amino acid sequence including QNLQEESAYF (SEQ ID NO: 16) on the amino acid sequence shown in SEQ ID NO: 2. can (specifically) bind to peptide fragments. That is, this CLEC4A function inhibiting antibody has the amino acid sequences QDFIFQNLQEESAYF (SEQ ID NO: 24), DFIFQNLQEESAYFV (SEQ ID NO: 25), FIFQNLQEESAYFVG (SEQ ID NO: 26), IFQNLQEESAYFVGL (SEQ ID NO: 27), FQNLQEESAYFVGLS (SEQ ID NO: 28), and QNLQEESAYFVGLSD (SEQ ID NO: 28). 29). The present invention provides a peptide fragment comprising or consisting of the amino acid sequence QNLQEESAYF (SEQ ID NO: 16), including, but not limited to, peptides of 10 to 20 amino acids in length, 10 to 15 amino acids in length, or 10 amino acids in length. ) provides a CLEC4A function-inhibiting antibody, which is an antibody that specifically binds to.

CLEC4A function-inhibiting antibodies (antibodies or antigen-binding fragments thereof) include peptide fragments (for example, but not limited to, 10 to 20 amino acids long, 10 ~15 amino acids long, or 10 amino acids long), for example, QNLQEESAYF (SEQ ID NO: 16) on the amino acid sequence shown in SEQ ID NO: 2. It may be one that can (specifically) bind to a peptide fragment consisting of the amino acid sequence FSSNCYFISTESASW (SEQ ID NO: 17). Such a CLEC4A function-inhibiting antibody is preferably an antibody that binds to a discontinuous epitope of the human CLEC4A protein, and in particular, an antibody that binds to an amino acid sequence ( For example, it is more preferable that the antibody binds (specifically) to a discontinuous epitope constituted by the amino acid sequence from position 113 to position 164 of SEQ ID NO: 2). Such a discontinuous epitope includes at least some amino acid residues of the amino acid sequences FSSNCYFISTESASW (SEQ ID NO: 17) and QNLQEESAYF (SEQ ID NO: 16) as a site recognized by the CLEC4A function-inhibiting antibody, and preferably includes It contains at least some amino acid residues of the sequence FSSNCYFISTESASW (SEQ ID NO: 17) and the entire amino acid sequence QNLQEESAYF (SEQ ID NO: 16). This CLEC4A function-inhibiting antibody may not bind to the peptide fragment consisting of SSNCYFISTESASWQ (SEQ ID NO: 30).

Alternatively, the CLEC4A function-inhibiting antibody (antibody or antigen-binding fragment thereof) may be a peptide fragment (for example, but not limited to, 10 to 20 amino acids long) comprising or consisting of the amino acid sequence QNLQEESAYF (SEQ ID NO: 16). , 10 to 15 amino acids long, or a 10 amino acid long peptide), for example, in addition to being able to bind to a peptide fragment consisting of a 15 amino acid long amino acid sequence including QNLQEESAYF (SEQ ID NO: 16) on the amino acid sequence shown in SEQ ID NO: 2. a peptide fragment (for example, but not limited to, a 14-20 amino acid long, 14-15 amino acid long, or 14 amino acid long peptide) comprising or consisting of the amino acid sequence SSNCYFISTESASW (SEQ ID NO: 18); For example, it may be one that can (specifically) bind to a peptide fragment consisting of a 15 amino acid long amino acid sequence including SSNCYFISTESASW (SEQ ID NO: 18) on the amino acid sequence shown by SEQ ID NO: 2. That is, the CLEC4A function-inhibiting antibody can bind to a peptide fragment consisting of the amino acid sequence FSSNCYFISTESASW (SEQ ID NO: 17) or SSNCYFISTESASWQ (SEQ ID NO: 30). Such a CLEC4A function-inhibiting antibody is preferably an antibody that binds to a discontinuous epitope of the human CLEC4A protein, and is an antibody that binds to an amino acid sequence (for example, More preferably, it is an antibody that (specifically) binds to a discontinuous epitope constituted by the amino acid sequence (114th to 164th amino acid sequence of SEQ ID NO: 2). Such a discontinuous epitope includes at least some amino acid residues of the amino acid sequences SSNCYFISTESASW (SEQ ID NO: 18) and QNLQEESAYF (SEQ ID NO: 16) as a site recognized by the CLEC4A function-inhibiting antibody, and preferably SSNCYFISTESASW ( It includes the entirety of SEQ ID NO: 18) and the entirety of QNLQEESAYF (SEQ ID NO: 16).

In yet another embodiment, the CLEC4A function-inhibiting antibody (antibody or antigen-binding fragment thereof) according to the present invention has an amino acid sequence of 15 amino acids that includes KKNMPVEETAWSCC (SEQ ID NO: 19) on the amino acid sequence shown in SEQ ID NO: 2. can (specifically) bind to the peptide fragment consisting of VKKNMPVEETAWSCC (SEQ ID NO: 31) and the peptide fragment consisting of KKNMPVEETAWSCCP (SEQ ID NO: 32). In a preferred embodiment, the CLEC4A function-inhibiting antibody specifically binds to an epitope comprising (e.g., consisting of) the amino acid sequence KKNMPVEETAWSCC (SEQ ID NO: 19) of the human CLEC4A protein, preferably a linear (continuous) epitope. . The present invention provides peptide fragments comprising or consisting of the amino acid sequence KKNMPVEETAWSCC (SEQ ID NO: 19), such as, but not limited to, peptides of 14-20 amino acids in length, 14-15 amino acids in length, or 14 amino acids in length. ) provides a CLEC4A function-inhibiting antibody, which is an antibody that specifically binds to.

Here, the term "peptide fragment" may refer to a peptide that is a fragment of a predetermined protein (in particular, full-length CLEC4A protein) or a synthetic peptide corresponding thereto. In the present invention, the term "peptide fragment" can be used interchangeably with the term "peptide." The peptide fragment or peptide in the present invention may be an oligopeptide. In the present invention, oligopeptide refers to a peptide having a length of 2 to 20 amino acids.

In this specification, amino acid residues and amino acid sequences are described according to the one-letter or three-letter notation commonly used in the field.

The CLEC4A function-inhibiting antibody according to the invention may be of any class of immunoglobulin molecules, such as IgG, IgE, IgM, IgA, IgD, or IgY, and further of any subclass, such as IgG1, IgG2, IgG3, IgG4, It may be IgA1, IgA2, etc.

The CLEC4A function-inhibiting antibody may be a monoclonal antibody or a polyclonal antibody. The CLEC4A function-inhibiting antibody may be in any antibody form, such as a multispecific antibody (such as a bispecific antibody), a recombinant antibody, a human antibody, a humanized antibody, a chimeric antibody, a single chain antibody, a mini antibody, etc. It may be a body, a diabody, a diabody, etc. The CLEC4A function-inhibiting antibody may also be a whole antibody or an antigen-binding fragment of a whole antibody, such as Fab, F(ab') ₂ , Fab', Fv, scFv, sdFv, and the like. The CLEC4A function-inhibiting antibody may be optionally modified such as glycosylation, acetylation, formylation, amidation, phosphorylation, or pegylation (PEG).

CLEC4A function-inhibiting antibodies can be produced by conventional methods. CLEC4A function-inhibiting antibodies can be produced based on known antibody production methods. For example, first, an antigen capable of inducing the production of CLEC4A function-inhibiting antibodies is administered subcutaneously, into the footpad, intraperitoneally, etc. to immunize the animal. Such antigens are antigens that include a target sequence (e.g., an epitope or a portion thereof) in the human CLEC4A protein to which the CLEC4A function-inhibiting antibody binds, including, but not limited to, full-length human CLEC4A protein, the above-mentioned A human CLEC4A protein fragment containing the target sequence, a polypeptide consisting of the above target sequence, an antigen containing the above target sequence (for example, a polypeptide consisting of the human CLEC4A protein partial sequence containing the above target sequence and other proteins such as immunoglobulin Fc molecules) fusion proteins with polypeptides), and cells that express polypeptides containing the above target sequence on their cell surfaces. The antigen may be administered with an optional adjuvant. The antigen may be administered once, but it is preferably administered multiple times (eg, 2 to 10 times, preferably 2 to 5 times). After immunization, the immune cells (eg, lymph node cells or spleen cells) are fused with known parent cells (eg, immortalized cells such as myeloma cells) by a conventional cell fusion method to obtain fused cells. The parent cell of the fusion partner is preferably derived from the same species as the immune cell. Mouse-derived myeloma cells used as parent cells for fusion include, but are limited to, P3U1 (P3-X63Ag8U1), P3 (P3x63Ag8.653), P3x63Ag8U.1, NS-1, MPC-11, etc. Not done.

Cell fusion between immune cells and parent cells can be carried out, for example, according to the method of Kohler and Milstein (Kohler, G. and Milstein, C. Methods Enzymol. (1981) 73, 3-46). Specifically, such cell fusion is carried out in normal nutrient broth in the presence of a cell fusion promoter. As the cell fusion promoter, for example, polyethylene glycol (PEG) having an average molecular weight of about 1000 to 6000 (eg, PEG4000), Sendai virus (HVJ), etc. can be used. Additional adjuvants such as dimethyl sulfoxide can also be used to increase fusion efficiency. The usage ratio of immune cells and parent cells (myeloma cells, etc.) can be set arbitrarily.

The obtained fused cells are screened for their ability to produce the desired CLEC4A function-inhibiting antibody and cloned to obtain monoclonal antibody-producing cells (hybridoma). A CLEC4A function-inhibiting antibody can be produced by collecting the antibody from the culture supernatant obtained by culturing the hybridoma.

When the CLEC4A function-inhibiting antibody is a polyclonal antibody, it can be obtained by collecting serum from an immunized animal (non-human animal) as described above. In this case, a non-human animal producing human antibodies can be used as the animal to be immunized.

Screening for CLEC4A function-inhibiting antibodies may be performed, for example, by examining the binding property to the antigen and/or peptide fragment to which the CLEC4A function-inhibiting antibody binds. For example, by detecting the binding of an antibody to the extracellular region of the human CLEC4A protein and/or the polypeptide consisting of the region consisting of the amino acid sequence shown in SEQ ID NO: 21 within the extracellular region of the human CLEC4A protein, CLEC4A function can be inhibited. Antibodies may also be screened.

In one embodiment, a peptide fragment consisting of the above-mentioned amino acid sequence WVDQTPYNESSTFW (SEQ ID NO: 15) and/or a peptide fragment consisting of one or both of the amino acid sequences QWVDQTPYNESSTFW (SEQ ID NO: 22) and WVDQTPYNESSTFWH (SEQ ID NO: 23), respectively. CLEC4A function-inhibiting antibodies can be screened by detecting the binding of antibodies to CLEC4A.

In another embodiment, a peptide fragment consisting of the above-mentioned amino acid sequence QNLQEESAYF (SEQ ID NO: 16) and/or QDFIFQNLQEESAYF (SEQ ID NO: 24), DFIFQNLQEESAYFV (SEQ ID NO: 25), FIFQNLQEESAYFVG (SEQ ID NO: 26), IFQNLQEESAYFVGL (SEQ ID NO: 26), 27), FQNLQEESAYFVGLS (SEQ ID NO: 28), and QNLQEESAYFVGLSD (SEQ ID NO: 29). CLEC4A function-inhibiting antibodies can be screened by detecting the binding of antibodies to peptide fragments consisting of each of the following. In addition to binding to the above-mentioned peptide fragment, CLEC4A function-inhibiting antibodies can also be screened by further detecting the binding of the antibody to the peptide fragment consisting of the above-mentioned amino acid sequence FSSNCYFISTESASW (SEQ ID NO: 17). Alternatively, in addition to binding to the above peptide fragment, a peptide fragment consisting of the above amino acid sequence SSNCYFISTESASW (SEQ ID NO: 18) and/or the amino acid sequence of one or both of FSSNCYFISTESASW (SEQ ID NO: 17) and SSNCYFISTESASWQ (SEQ ID NO: 30) It is also possible to screen for antibodies that inhibit CLEC4A function by detecting the binding of antibodies to peptide fragments consisting of each of the following.

In yet another embodiment, a peptide fragment consisting of the above-described amino acid sequence KKNMPVEETAWSCC (SEQ ID NO: 19) and/or consisting of one or both of the amino acid sequences VKKNMPVEETAWSCC (SEQ ID NO: 31) and KKNMPVEETAWSCCP (SEQ ID NO: 32), respectively. CLEC4A function-inhibiting antibodies can also be screened by detecting antibody binding to peptide fragments.

Binding of antibodies to peptide fragments can be detected by any method capable of detecting an antigen-antibody reaction; for example, it may be detected using a peptide array on which target peptide fragments are immobilized. .

CLEC4A function-inhibiting antibodies can also be produced as recombinant antibodies by cloning the antibody gene, inserting it into an appropriate vector, introducing this into a host, and producing it using genetic recombination technology. . Specifically, the cDNA of the antibody variable region (V region) is ligated with the DNA encoding the antibody constant region (C region), and is inserted under the control of the expression control region of an expression vector, such as an enhancer or promoter. A CLEC4A function-inhibiting antibody can be produced as a recombinant antibody by introducing an expression vector into a host cell, transforming it, and causing the host cell to produce the antibody.

Antibodies, nucleic acids, etc. obtained as described above can be isolated and purified by known purification methods and recovered. Purification methods include affinity purification, reversed-phase chromatography, reversed-phase high performance liquid chromatography (RP-HPLC), ion exchange chromatography and other chromatography methods, gel filtration, column purification, and polyacrylamide gel electrophoresis (PAGE). etc., or any combination thereof, but is not limited to these.

The CLEC4A function-inhibiting antibody according to the present invention has an immune checkpoint inhibiting effect. CLEC4A function-inhibiting antibodies cause immune checkpoint inhibition in antigen-presenting cells such as dendritic cells by inhibiting CLEC4A function, release the suppression of anti-tumor immune responses (cancer immune responses) contributed by immune checkpoint molecules, and produce anti-tumor immune responses. It can activate tumor immune response and enhance effective anti-tumor immune response (cancer immune response).

Here, the antigen-presenting cells in which immune checkpoint inhibition is caused by the CLEC4A function-inhibiting antibody according to the present invention are antigen-presenting cells that express CLEC4A protein on the cell surface, and such antigen-presenting cells include , dendritic cells, B cells (eg, CD19 ⁺ B cells), monocytes (eg, CD14 ⁺ monocytes), and the like, but are not limited to these. Note that antigen-presenting cells are cells that present antigens on the cell surface to activate T cells. Therefore, the CLEC4A function-inhibiting antibody according to the present invention is an immune checkpoint inhibitor, preferably directed against antigen-presenting cells such as dendritic cells, B cells (e.g., CD19 ⁺ B cells), and monocytes (e.g., CD14 ⁺ monocytes). It can be used as an immune checkpoint inhibitor.

The immune checkpoint inhibitory effect (immune checkpoint inhibitory activity) and the ability to activate anti-tumor immune responses of the CLEC4A function-inhibiting antibody of the present invention can be observed in antigen-presenting cells, such as dendritic cells, treated with the CLEC4A function-inhibiting antibody. Increased production of inflammatory cytokines (e.g., IL-6, TNF-α, IL-12p40, etc.) compared to when not treated with a CLEC4A function-inhibiting antibody (when a control antibody that does not inhibit CLEC4A function is administered) (preferably a statistically significant increase) can be used as an index for evaluation. More specifically, the evaluation of the immune checkpoint inhibiting effect and the ability to activate antitumor immune responses of CLEC4A function-inhibiting antibodies is carried out by targeting antigen-presenting cells, preferably dendritic cells, to TLR ligands such as TLR4 ligands and TLR9 ligands, For example, this can be carried out by culturing in the presence of LPS or CpG-B ODN 1668, etc., adding a CLEC4A function-inhibiting antibody, measuring the amount of cytokine in the culture solution, and comparing it with a control. If the production amount of inflammatory cytokines (e.g., IL-6, TNF-α, IL-12p40, etc.) is increased (preferably statistically significantly increased) compared to the control, the antibody may be detected as an immune checkpoint. It can be judged that it has an inhibitory effect (immune checkpoint inhibitory activity) and an ability to activate an antitumor immune response.

The CLEC4A function-inhibiting antibody according to the present invention can be used as an active ingredient of an antitumor drug. The present invention also provides an antitumor drug or a pharmaceutical composition comprising the CLEC4A function-inhibiting antibody according to the present invention. Preferably, the antitumor drug or pharmaceutical composition contains the CLEC4A function-inhibiting antibody according to the present invention in a therapeutically effective amount. Such an antitumor drug or pharmaceutical composition may be used for cancer treatment or prevention, for example, for suppressing cancer progression or preventing cancer recurrence. The antitumor drug or pharmaceutical composition according to the present invention provides cancer treatment based on immune checkpoint inhibition of antigen-presenting cells such as dendritic cells, B cells (CD19 ⁺ B cells), and monocytes (CD14 ⁺ monocytes). Or it is useful for prevention, for example, suppressing cancer progression or preventing cancer recurrence.

In the present invention, "suppressing the progression of cancer" includes delaying or preventing the deterioration of the cancer condition (in particular, increase in tumor size), or reversing the disease condition. In one embodiment, inhibition of cancer progression can be evaluated using tumor size (eg, tumor volume) as an index. For example, tumor size (e.g., tumor volume) is measured (preferably over time) in a subject (cancer patient) or tumor-bearing animal model to which the CLEC4A function-inhibiting antibody of the present invention has been administered, and If a reduction in tumor size (preferably a statistically significant reduction) is observed compared to administering an antibody that does not have antitumor activity in place of the CLEC4A function-inhibiting antibody according to the invention, cancer It can be judged that the progress has been suppressed.

Cancers or tumors targeted for activation of anti-tumor immune responses and treatment or prevention by the CLEC4A function-inhibiting antibody according to the present invention include, but are not limited to, malignant melanoma, lung cancer, and renal cells. Head and neck cancer, bladder cancer, pancreatic cancer, stomach cancer, liver cancer, esophageal cancer, biliary tract cancer, colorectal cancer, urothelial cancer, glioblastoma, multiple myeloma, and ovarian cancer. Cervical cancer, endometrial cancer, breast cancer, malignant pleural mesothelioma, soft tissue sarcoma, lymphoma (Hodgkin lymphoma, primary central nervous system lymphoma, primary testicular lymphoma, etc.), virus-positive or negative solid tumors, Merkel cell Examples include cancer.

The antitumor drug or pharmaceutical composition according to the present invention may further contain a pharmaceutically acceptable additive in addition to the CLEC4A function-inhibiting antibody. Such additives include, but are not limited to, carriers, suspending agents, binders, excipients, lubricants, disintegrants, wetting agents, stabilizers, buffers, preservatives, colorants, pH Conditioners and the like can be mentioned. The additives can be used alone or in combination of two or more, and can be used as appropriate depending on the dosage form of the preparation.

The CLEC4A function-inhibiting antibody of the present invention, or the antitumor drug or pharmaceutical composition containing the same, can be administered by any administration route such as parenteral or oral administration, but parenteral administration is preferable, such as intravenous administration. It can be administered intraarterially, intramuscularly, intraperitoneally, intrathecally, epidurally, subcutaneously, intradermally, nasally, pulmonaryly, or intratumorally. The CLEC4A function-inhibiting antibody of the present invention, or an antitumor drug or pharmaceutical composition containing the same, may be administered systemically or locally.

The present invention also provides a method for inhibiting immune checkpoints, which comprises administering to a subject (patient) the CLEC4A function-inhibiting antibody according to the present invention, or an antitumor drug or pharmaceutical composition containing the same. The present invention also provides a method for activating an antitumor immune response (or Methods for inducing anti-tumor immune responses) are also provided. The present invention also provides a method for treating or preventing cancer, which comprises administering to a subject a CLEC4A function-inhibiting antibody, or an antitumor drug or pharmaceutical composition containing the same. The method for treating or preventing cancer may be a method for inhibiting cancer progression or a method for preventing cancer recurrence.

The target is preferably a mammal, such as humans, primates such as chimpanzees and gorillas, rodents such as mice, rats, and guinea pigs, cows, horses, pigs, sheep, goats, llamas, camels, dogs, and cats. , a mammal such as a rabbit, but preferably a human. The subject may be a subject who has developed or is suspected of having immunosuppression due to immune checkpoint molecules. The subject typically may be a subject who has cancer or is suspected of having cancer. The subject may be a subject who has become refractory to cancer immunotherapy or has decreased responsiveness to cancer immunotherapy. Refractoriness, or decreased responsiveness, to such cancer immunotherapies may be associated with immune checkpoints, particularly dendritic cells, B cells (e.g., CD19 ⁺ B cells), monocytes (e.g., CD14 ⁺ monocytes). ), etc., may be caused by immune checkpoints in antigen-presenting cells. The subject may also be a patient suffering from cancer that is refractory to or resistant to inhibitors of immune checkpoint molecules (CTLA-4, PD-1, PD-L1, etc.) expressed on T cells. The subject may be a cancer patient who is refractory or resistant to existing immune checkpoint inhibitors. Alternatively, the subject has cancer that is refractory or intolerant to immune checkpoint inhibitors (e.g., inhibitors against immune checkpoint molecules expressed on T cells; hereinafter also referred to as T cell-expressed immune checkpoint molecule inhibitors). It may be a patient.

The administration method can be appropriately selected by those skilled in the art depending on the species, age, weight, sex, symptoms, etc. of the subject (patient). The dosage of the CLEC4A function-inhibiting antibody can be set, for example, in the range of 0.0001 mg to 1000 mg per kg of body weight at a time, and/or in the range of 0.001 mg to 100000 mg per subject. is not limited to the range of

Administration of a CLEC4A function-inhibiting antibody, or an antitumor drug or pharmaceutical composition containing the same, to a subject enhances the production of inflammatory cytokines in antigen-presenting cells, such as dendritic cells, which stimulate T cells and other immune It activates/enhances cell functions and activates (induces) anti-tumor immune responses, resulting in effects such as suppressing cancer progression, tumor regression, or preventing cancer recurrence in the subject. In a preferred embodiment, the CLEC4A function-inhibiting antibody of the present invention, or an antitumor drug or pharmaceutical composition containing the same, (i) inhibits the induction and/or accumulation of myeloid-derived suppressor cells in lymphoid tissue and/or cancer tissue. (ii) induction and/or accumulation of effector T cells, tumor-infiltrating T cells (e.g., CD4 ⁺ T cells and CD8 ⁺ T cells), and activated dendritic cells in lymphoid and/or cancer tissues; (iii) suppression of immune tolerance in the cancer microenvironment; and (iv) activation of an anti-tumor immune response.

Cancer cells that manifest in vivo are thought to escape from immune surveillance mechanisms, and immune checkpoint molecules are said to be one of the major escape mechanisms. In recent years, immune checkpoint inhibitors (anti-CTLA-4 antibody [ipilimumab; Yervoy], anti-PD-1 antibody [nivolumab; Opdivo], anti-PD-L1 antibody [avelumab; Bavencio], etc.) that have been clinically applied have certain Although it has been shown to be highly effective against various cancers, there are concerns about non-responsiveness and acquired resistance to various cancers, as well as the development of immune-related adverse events (irAEs). In contrast, the CLEC4A function-inhibiting antibody of the present invention is effective against immune-related adverse events (e.g., autoimmune pathologies such as autoimmune diseases or disorders, weight loss, tissue damage, etc.), particularly when immune checkpoint molecules expressed on T cells are suppressed. High antitumor effects can be brought about while reducing the risk of immune-related adverse events caused by inhibition. Therefore, the CLEC4A function-inhibiting antibody of the present invention, or an antitumor drug or pharmaceutical composition containing the same, can achieve both high antitumor effects and higher safety.

In the present invention, the above-described CLEC4A function-inhibiting antibody, or an antitumor drug or pharmaceutical composition containing the same, may be used in combination (concomitant administration) with other immune checkpoint inhibitors. Other immune checkpoint inhibitors typically include, but are not limited to, inhibitors against immune checkpoint molecules expressed on T cells. Examples of immune checkpoint molecules expressed on T cells include, but are not limited to, PD-1, CTLA-4, PD-L1, Tim-3, BTLA, LAG-3, TIGHT, and the like. An inhibitor for immune checkpoint molecules is any substance that has the effect of inhibiting the function of the immune checkpoint molecule (protein) itself or the function of the gene encoding the immune checkpoint molecule (antagonist effect), such as an antibody ( Preferably, it may be a neutralizing antibody) or a nucleic acid molecule that inhibits the expression of an immune checkpoint molecule (expression of a gene encoding the molecule). Such inhibitory nucleic acid molecules may be, but are not limited to, siRNA (small interfering RNA), shRNA (short hairpin RNA), hpRNA (hairpin RNA), or longer nucleic acids including . In one embodiment, the inhibitor against an immune checkpoint molecule expressed on T cells (T cell expressed immune checkpoint molecule inhibitor) may be an existing immune checkpoint inhibitor, such as an anti-CTLA-4 antibody. (ipilimumab, etc.), anti-PD-1 antibodies (nivolumab, etc.), anti-PD-L1 antibodies (avelumab, etc.), etc. may be used. The above-mentioned CLEC4A function-inhibiting antibody, or an antitumor drug or pharmaceutical composition containing the same, can be used in combination with, for example, a T cell-expressed immune checkpoint molecule inhibitor, compared to the case where the above-mentioned CLEC4A function-inhibiting antibody is used alone. However, it can bring about significantly higher and synergistic cancer treatment effects. Therefore, the present invention also provides the above-mentioned CLEC4A function-inhibiting antibody, or an antitumor drug or pharmaceutical composition containing the same, for use in combination with a T cell-expressed immune checkpoint molecule inhibitor. Furthermore, the present invention also provides an antitumor drug or a pharmaceutical composition containing an inhibitor of T cell-expressed immune checkpoint molecules in addition to the above-mentioned CLEC4A function-inhibiting antibody. Such an antitumor drug or pharmaceutical composition preferably contains the above-mentioned CLEC4A function-inhibiting antibody and T cell-expressed immune checkpoint molecule inhibitor, each in a therapeutically effective amount. Subjects to whom the above-mentioned antitumor drug or pharmaceutical composition, or combination therapy that uses a combination of the CLEC4A function-inhibiting antibody and T cell-expressed immune checkpoint molecule inhibitor, should be applied (administered) to existing immune checkpoint inhibitors. The patient may be a cancer patient who is responsive or resistant to the disease, or a cancer patient who is refractory or non-resistant to immune checkpoint inhibitors (eg, T cell-expressed immune checkpoint molecule inhibitors). In combined administration, the above-mentioned CLEC4A function-inhibiting antibody, or an antitumor drug or pharmaceutical composition containing it, and another immune checkpoint inhibitor (T cell-expressed immune checkpoint molecule inhibitor, etc.) are administered at any time. For example, they may be administered simultaneously, sequentially, or at separate times, and may be administered as separate agents or in a single agent.

Administration of the above-mentioned CLEC4A function-inhibiting antibody or an antitumor drug or pharmaceutical composition containing the same to a subject can suppress the expression of immunosuppressive molecules in cancer tissues. The above CLEC4A function-inhibiting antibodies can, for example, suppress the expression of IL (interleukin)-10, IDO (indoleamine 2,3-dioxygenase), arginase, and mPGES (membrane-associated prostaglandin E2 synthase). can. The combined administration of the above-mentioned CLEC4A function-inhibiting antibody and T cell-expressed immune checkpoint molecule inhibitor can more strongly suppress the expression of immunosuppressive molecules in cancer tissues, such as IL-10, IDO, arginase, The expression of at least one selected from the group consisting of COX-2, mPGES, TGF (transforming growth factor)-β, and iNOS (inducible nitric oxide synthase) can be significantly suppressed.

The present invention provides a method for activating an antitumor immune response or a method for treating or preventing cancer, including the administration of the above-mentioned CLEC4A function-inhibiting antibody or an antitumor drug or pharmaceutical composition containing the same. May be used in conjunction with vaccine therapy. Examples of combination cancer vaccine therapies include administering to a patient a cancer antigen, a combination of a cancer antigen and an agonist TLR ligand, or a combination of a cancer antigen, an agonist TLR ligand, and an agonist anti-CD40 antibody. . Examples of cancer antigens include WT1, MAGE-A1, MAGE-A3, gp100, Melan A, NY-ESO-1, MUC1, LMP2, HPV E6 E7, EGFRvIII, HER-2/neu, idiotype, p53 non- Examples include, but are not limited to, mutant (p53 nonmutant), BAGE, tyrosinase, CEA, bcr/abl, ras, and the like. Agonist TLR ligands (also called agonistic TLR ligands or TLR agonists) include agonist ligands such as TLR1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and/or 13; Examples include agonist TLR9 ligands that are unmethylated CpG dinucleotide motif-containing oligonucleotides such as CpG ODN 2006 and CpG-B ODN 1668. Agonist TLR ligands suitable for administration to human subjects can be agonist ligands for human TLR1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

The present invention provides a method for activating an antitumor immune response or a method for treating or preventing cancer, including the administration of the above-mentioned CLEC4A function-inhibiting antibody or an antitumor drug or pharmaceutical composition containing the same. It may be used in conjunction with standard treatments (surgery for tumor removal, radiation therapy, chemotherapy).

Hereinafter, the present invention will be explained in more detail using Examples. However, the technical scope of the present invention is not limited to these Examples.

In addition, the flow cytometry method in the following examples was implemented as follows.
<Flow cytometry method>
The following fluorescently labeled monoclonal antibodies were used to stain cells in flow cytometry analysis.

Anti-mouse CD3ε antibody (clone 145-2C11; BD Biosciences), anti-mouse CD4 antibody (clone RM4-5; BD Biosciences), anti-mouse CD8α antibody (clone 53.6.7; BD Biosciences), anti-mouse CD11b antibody (clone M1/ 70; Biolegend), anti-mouse CD11c antibody (clone HL3; BD Biosciences), anti-mouse CD40 antibody (clone 3/23; BD Biosciences), anti-mouse CD44 antibody (clone IM7; BD Biosciences), anti-mouse CD80 antibody (clone 16) -10A1; BD Biosciences), anti-mouse CD86 antibody (clone GL1; BD Biosciences), anti-mouse CD62L antibody (clone MEL-14; Biolegend), anti-mouse B220 antibody (clone RA3-6B2; BD Biosciences), anti-mouse F4/ 80 antibody (clone BM8; Biolegend), anti-mouse NK1.1 antibody (clone PK136; BD Biosciences), anti-mouse MHC class I antibody (clone AF6-88.5; BD Biosciences), anti-mouse MHC class II antibody (clone M5/114.15) .2; BD Biosciences), anti-mouse B7-H1 antibody (clone MIH5; eBioscience), anti-mouse B7-DC antibody (clone TY25; eBioscience), anti-mouse B7-H2 antibody (clone HK5.3; eBioscience), anti-mouse SiglecH antibody (clone 551; Biolegend), anti-mouse Gr-1 antibody (clone RB6-8C5; Biolegend), anti-mouse Clec4A4 antibody (clone 33D1; Biolegend), anti-mouse IFN-γ antibody (clone XMG1.2; BD Biosciences) , anti-mouse CD19 antibody (clone 6D5; Biolegend), or control rat IgG antibody (BD Biosciences).

Anti-human CD3 antibody (clone UCHT1; TOMBO), CD8α (clone SK1; BD Biosciences), anti-human CD11c antibody (clone 3.9; TOMBO), anti-human CD14 antibody (clone 61D3; TOMBO), anti-human CD19 antibody (clone HIB19; TOMBO), anti-human CD56 antibody (clone MY31; TOMBO), anti-human CD1c antibody (clone L161; Biolegend), anti-human CD40 antibody (clone 5C3; BD Biosciences), anti-human CD80 antibody (clone L307.4; BD Biosciences) , anti-human CD86 antibody (clone IT2.2; BD Biosciences), anti-human CD141 antibody (clone M80; Biolegend), anti-human CD303 antibody (clone 201A; Biolegend), anti-human HLA-DR antibody (clone L243; Biolegend), Anti-human CLEC4A antibody (clone 9E8; Biolegend) or control mouse IgG antibody (BD Biosciences).

Cell surface molecules were stained by incubating cells (1-5 x 10 ⁵ cells) with fluorescently labeled monoclonal antibodies for 30 minutes at 4°C.

In some flow cytometry analyses, the following fluorescently labeled MHC-peptide complexes were used to stain cells instead of fluorescently labeled monoclonal antibodies: Mouse H-2Kb OVA peptide pentamer.

Fluorescent staining analysis was performed using a FACSVerse ^™ flow cytometer (BD Biosciences) and FlowJo software (Tree star).

Moreover, statistical analysis was performed as follows regarding the data obtained in the following examples.
<Statistical analysis>
Analysis of variance (ANOVA) was used to analyze statistically significant differences in the data. A P value of less than 0.01 was considered significant. Data in the figure is shown as an average value (±standard deviation).

[Example 1]
1) Mouse In this example, C57BL/6 mice (Japan Clea) were used as wild type (WT) mice. Clec4A4-deficient mice (Clec4a4 ^- / ^- mice) were produced using C57BL/6 mice according to the description in Non-Patent Document 4. Clec4A4-deficient mice (strain name B6.Cg-Clec4a4<tm1.1Ksat>) have been deposited and available at the RIKEN BioResource Center (RIKEN BRC; Japan) under accession number RBRC09657. Note that Clec4A4 is a mouse ortholog of CLEC4A.

2) Immune response after cancer vaccination in non-tumor-bearing mouse model
Ovalbumin (OVA) (500 μg/mouse; Sigma-Aldrich) and CpG-B ODN, a Toll-like receptor (TLR) 9 ligand, were administered to WT mice and Clec4A4-deficient mice as cancer vaccines. 1668 (50 μg/mouse; Hokkaido System Science) and agonist anti-CD40 antibody (10 μg/mouse; clone 1C10; Biolegend) were administered intraperitoneally for immunization. On day 6 post-immunization, we induced OVA-specific cytotoxic T cells (CTLs) (CD44 ^high OVA-MHC class I pentamer-binding CD8 ⁺ T cells) and OVA-specific IFN-γ-producing CD8 ⁺ T cells in the spleen. , analyzed by the flow cytometry method described above. As a control, similar cell analysis was performed on the spleens of unvaccinated WT mice and Clec4A4-deficient mice.

In WT mice, antigen-specific CTL CD44 ^high OVA-MHC class I pentamer-bound CD8 ⁺ It was shown that the induction of T cells (FIGS. 1A and B) and antigen-specific IFN-γ producing CD8 ⁺ T cells (FIGS. 2A and B) was enhanced. On the other hand, in the cancer vaccine immunized group of Clec4A4-deficient mice, not only were antigen-specific CTL and antigen-specific IFN-γ-producing CD8 ⁺ T cells induced, but compared to the cancer vaccine immunized group of WT mice, The induction of antigen-specific CTL and antigen-specific IFN-γ-producing CD8 ⁺ T cells was significantly enhanced (Figures 1 and 2).

3) Evaluation of cancer progression in tumor-bearing mouse models and immune responses after cancer vaccination
Tumor-bearing mice were generated by subcutaneously implanting (1x105 cells/mouse) a recombinant melanoma cell line expressing ovalbumin (OVA) (B16-OVA melanoma; Brown DM, et al., Immunology. (2001) 102:486-497) into the dorsum of wild-type and ^Clec4A4 -deficient mice. Tumor volume was measured daily using a digital caliper (Digimatic Caliper, Mitutoyo) until 23 days after tumor implantation, and tumor progression was evaluated.

Tumor-bearing WT mice and tumor-bearing Clec4A4-deficient mice were immunized with a cancer vaccine by intraperitoneal administration 7 days after cancer cell transplantation. Thirteen days after cancer cell transplantation, the induction of CD44 ^high OVA-MHC class I pentamer-binding CD8 ⁺ T cells, which are OVA-specific CTLs, and OVA-specific IFN-γ-producing CD8 ⁺ T cells was determined by flow cytometry as described above. It was analyzed using the method.

In tumor-bearing WT mice and tumor-bearing Clec4A4-deficient mice, antigen-specific CTL and antigen-specific IFN-γ production in the cancer vaccine immunized group was significantly lower than in non-tumor-bearing WT mice and Clec4A4-deficient mice. Induction of CD8 ⁺ T cells was enhanced (Figures 1 and 2). Furthermore, the induction of antigen-specific CTL and antigen-specific IFN-γ-producing CD8 ⁺ T cells in the cancer vaccine-immunized group of tumor-bearing Clec4A4-deficient mice compared to the cancer-vaccine immunized group of tumor-bearing WT mice. was significantly enhanced (Figures 1 and 2).

From 15 days after cancer cell transplantation, tumor-bearing Clec4A4-deficient mice (non-vaccinated) showed no immune-related adverse events and developed cancer resistance compared to tumor-bearing WT mice (non-vaccinated). Progression was significantly inhibited (Fig. 3A-C). On the other hand, in the cancer vaccine-immunized group of tumor-bearing WT mice, tumor regression was clearly observed 15 days after cancer cell transplantation. Furthermore, in the cancer vaccine immunized group of tumor-bearing Clec4A4-deficient mice, compared to the cancer vaccine immunized group of tumor-bearing WT mice, the effect of the cancer vaccine on suppressing cancer progression was significantly lower 23 days after cancer cell transplantation. An increase in the number of patients was observed (Figure 3).

These results demonstrate that mouse Clec4A4 is an immune checkpoint molecule expressed on dendritic cells and acts as a brake on antitumor immune responses.

4) Immune response in tumor-bearing mouse model
A recombinant malignant melanoma cell line expressing ovalbumin (OVA) (B16-OVA melanoma; Brown DM, et al., Immunology. (2001) 102:486-497) was found in the backs of WT mice and Clec4A4-deficient mice. Tumor-bearing mice were produced by subcutaneously transplanting (1x10 ⁵ cells/mouse). Twenty days after cancer cell transplantation, the induction of various immune cells in the spleens and cancer tissues of tumor-bearing WT mice and cancer-bearing Clec4A4-deficient mice was analyzed by the flow cytometry method described above. Spleen mononuclear cells from non-tumor-bearing mice and tumor-bearing mice were prepared from spleen cells using red blood cell lysis buffer (Red Blood Cell Lysing Buffer Hybri-Max ^™ ; Sigma-Aldrich) (Non-Patent Document 4). . Spleen mononuclear cells and cancer tissues from tumor-bearing mice were prepared 20 days after cancer cell transplantation. As controls, non-tumor-bearing WT mice and non-tumor-bearing Clec4A4-deficient mice were similarly analyzed by flow cytometry. The results of the analysis are shown in Figures 4 to 8.

Compared to tumor ^- bearing WT mice, tumor-bearing Clec4A4-deficient mice showed ^increased myeloid-derived suppressor cells in the spleen ( ^Figure 4A) and cancer tissues (Figure 4B). Suppression of the increase in suppressor cells (MDSCs) was observed.

In addition, compared to tumor-bearing WT mice, cancer-bearing Clec4A4-deficient mice showed increased levels of CD11c ⁺ SiglecH ^- cDCs (normal dendritic cells) and CD11c ^low SiglecH ⁺ pDCs (plasmacytoid dendritic cells) in cancer tissues. Enhanced accumulation was observed (Fig. 4B).

Furthermore, compared to tumor-bearing WT mice, tumor-infiltrating CD4 ⁺ /CD8 ⁺ lymphocytes (TILs) and NK1.1 ⁺ Increased accumulation of NK cells was observed (Figure 4B).

In tumor-bearing WT mice, an increase in Gr1-1 ⁺ CD11b ⁺ F4/80 ⁺ MDSCs in the spleen was observed compared to non-tumor-bearing WT mice (Fig. 4A), and a marked increase in cancer tissue. Accumulation was observed (Fig. 4B).

In tumor-bearing WT mice, an increase in CD44 ⁺ CD62L ^- effector CD4 ⁺ T cells in the spleen was observed compared with non-tumor-bearing WT mice (Fig. 5A), but the increase in CD8 ⁺ T cells was It was not observed (Fig. 5B). On the other hand, in tumor-bearing Clec4A4-deficient mice, CD44 ⁺ CD62L ^-effector CD4 ⁺ T cells/CD8 ⁺ T cells increased in the spleen (Figure 5) and cancer tissues (Figure 6) compared to tumor-bearing WT mice. were observed (A: CD4 ⁺ T cells, B: CD8 ⁺ T cells).

In tumor-bearing WT mice, decreased expression of MHC I, CD40, CD80, B7-H1, and B7-H2 in cDCs in the spleen was observed compared to non-tumor-bearing WT mice (FIG. 7). On the other hand, in tumor-bearing Clec4A4-deficient mice, enhanced expression of MHC I, CD80, CD86, B7-H1, and B7-H2 in cDCs in the spleen was observed compared to non-tumor-bearing Clec4A4-deficient mice ( Figure 7).

Furthermore, compared to tumor-bearing WT mice, enhanced expression of MHC class I (MHC I), CD80, CD86, B7-H1, and B7-H2 in splenic cDCs was observed in tumor-bearing Clec4A4-deficient mice ( Figure 7).

Furthermore, compared to tumor-bearing WT mice, tumor-bearing Clec4A4-deficient mice showed enhanced expression of MHC I, MHC class II (MHC II), CD80, CD86, and B7-H1 in tumor-infiltrating cDCs, and increased expression of B7-H2 and A decrease in the expression of B7-DC was observed (FIG. 8).

In tumor-bearing Clec4A4-deficient mice, compared to tumor-bearing WT mice, the expression of MHC I, MHC II, CD80, CD86, and B7-H1 in cDCs was enhanced in cancer tissues, and the expression of B7-H2 and B7-DC was increased. A decrease in expression was observed (Fig. 8).

From the above analysis results, inhibition of dendritic cell function by Clec4A4 promotes the induction and accumulation of myeloid-derived suppressor cells, as well as effector T cells, tumor-infiltrating T cells, and activated dendritic cells in lymphoid tissues and cancer tissues. It was shown that cancer progression was promoted by inducing immune tolerogenicity in the cancer microenvironment and suppressing anti-tumor immune responses based on the induction and suppression of accumulation of ). The above analysis results indicate that inhibition of Clec4A4 function leads to activation of anti-tumor immune response and cancer suppression.

Furthermore, in mice, Clec4A4 deficiency enhances cancer immune responses and suppresses cancer progression. ” was proven.

[Example 2]
Peripheral blood mononuclear cells were separated from human peripheral blood (peripheral blood of a healthy individual) by specific gravity centrifugation using Ficoll-Paque ^™ PLUS (GE Healthcare Life Sciences). Peripheral blood monocytes were purified from the separated peripheral blood mononuclear cell fraction using Monocyte Isolation Kit II (Miltenyi Biotec) and cell separation device autoMACS ^(R) Pro Separator (Miltenyi Biotec). .

According to the method described in Non-Patent Document 3, the obtained human peripheral blood monocytes were treated with human recombinant granulocyte-macrophage colony stimulating factor (GM-CSF) (50 ng/ml, Wako) and human recombinant IL-4 (100 ng). Human peripheral blood monocyte-derived dendritic cells were produced by culturing them for one week in the presence of 1/ml, Wako). In addition, human peripheral blood monocytes obtained according to the method described in Non-Patent Document 3 were treated with human recombinant GM-CSF (50 ng/ml, Wako), human recombinant IL-4 (100 ng/ml, Wako), By culturing for 1 week in the presence of human recombinant IL-10 (50 ng/ml, Wako) and human recombinant TGF-β1 (50 ng/ml, Wako), human peripheral blood monocyte-derived immunosuppressive Dendritic cells were produced. Peripheral blood monocyte-derived immunosuppressive dendritic cells are known to be generated in the cancer environment.

Expression of CLEC4A in CD3 ⁺ T cells, CD19 ⁺ B cells, CD14 ⁺ monocytes, CD11c ⁺ dendritic cells, and CD56 ⁺ natural killer (NK) cells in peripheral blood mononuclear cell fractions obtained from healthy subjects. Analysis was performed using the flow cytometry method described above. The expression of the cell surface molecule (CLEC4A) in each cell is shown in a histogram in FIG. 9A.

In the peripheral blood mononuclear cell fraction, CLEC4A expression was not observed in CD3 ⁺ T cells or CD56 ⁺ NK cells, but was observed in CD19 ⁺ B cells, CD14 ⁺ monocytes, and CD11c ⁺ dendritic cells (FIG. 9A). Furthermore, CD14 ⁺ monocytes and CD11c ⁺ dendritic cells showed higher expression for CLEC4A compared to CD19 ⁺ B cells (Figure 9A).

Analysis of the expression of CD11c, HLA-DR, and CLEC4A in CD141 ⁺ cDC1 cells, CD1c ⁺ cDC2 cells, and CD303 ⁺ pDC cells in the peripheral blood mononuclear cell fraction obtained from healthy individuals using the flow cytometry method described above. did. The expression of each cell surface molecule (CD141, CD1c, CD303, CD11c, HLA-DR, and CLEC4A) is shown in a histogram in FIG. 9B.

In peripheral blood dendritic cell subpopulations in the peripheral blood mononuclear cell fraction from healthy individuals, CD1c ⁺ CD11c ⁺ HLA-DR ⁺ cDC2 cells, CD141 ⁺ CD11c ⁺ HLA-DR ⁺ cDC1 cells, and CD303 ⁺ CD11c ^- showed higher expression for CLEC4A compared to HLA-DR ⁺ pDC cells (Figure 9B). Note that CD1c ⁺ peripheral blood mononuclear cells obtained by flow cytometry analysis were designated as cDC2, CD141 ⁺ peripheral blood mononuclear cells as cDC1, and CD303 ⁺ peripheral blood mononuclear cells as pDC.

Furthermore, peripheral blood monocytes obtained from healthy individuals, prepared peripheral blood monocyte-derived dendritic cells (in the figure, monocyte-derived DC), and peripheral blood monocyte-derived immunosuppressive dendritic cells (in the figure, The expression of CD11c, CD40, CD80, CD86, HLA-DR, and CLEC4A in monocyte-derived DCreq was analyzed by the flow cytometry method described above. The expression of each cell surface molecule is shown in a histogram in FIG. 9C.

Peripheral blood monocyte-derived immunosuppressive dendritic cells showed lower expression of CD11c, CD86, and HLA-DR compared to peripheral blood monocyte-derived dendritic cells, but equally high expression of CLEC4A. (Fig. 9C).

In mice, Clec4A4 expression is limited to CD1c ⁺ peripheral blood mononuclear cells (cDC2) (Non-Patent Document 4), but in humans, cDC2 is expressed in CD141 ⁺ peripheral blood mononuclear cells (cDC1) and CD303 ⁺ peripheral blood mononuclear cells. In addition to showing higher CLEC4A expression than pDCs, CLEC4A expression was also observed in monocytes and B cells. Furthermore, CLEC4A expression is also observed in immunosuppressive dendritic cells generated in the cancer environment (Non-Patent Document 3 and Nagayama H., et al., Melanoma Res., (2003) 13, pp.521-530). was recognized. These results suggested that in humans, CLEC4A is a functional regulatory molecule expressed in antigen-presenting cells.

[Example 3]
A BamHI recognition sequence (5'-ggatcc- cDNA (SEQ ID NO: 11) with an XhoI recognition sequence (5'-ctcgag-3') added to the 3' end was synthesized using GeneArt ^(R) (Life Technologies).

Similarly, human CLEC4A ITIM (Immunoreceptor tyrosine-based Inhibition motif) sequence deletion mutant (SEQ ID NO: 4; ΔI5-V10; 231 amino acid length), CLEC4A extracellular region deletion mutant (SEQ ID NO: 6; ΔF69-L237; 68 amino acids long), CLEC4A carbohydrate-recognition domain (CRD)-EPS deletion mutant (SEQ ID NO: 8; ΔE195-S197; 234 amino acids long), and CLEC4A A BamHI recognition sequence is located at the 5' end of the base sequence (SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, and SEQ ID NO: 9, respectively) encoding the sugar chain modification site substitution variant (SEQ ID NO: 10; N185Q; 237 amino acids long). , cDNA with an XhoI recognition sequence added to the 3' end in the same manner as above was synthesized using GeneArt ^(R) (Life Technologies).

Next, these cDNAs were treated with restriction enzymes BamHI and XhoI and introduced into the BamHI-XhoI site in the multiple cloning site of the pMX-IRES-GFP retroviral vector to produce a CLEC4A or mutant expression retroviral vector. Additionally, retroviral packaging cells (Phoenix) were infected with these retroviral vectors or a control retroviral vector (not containing CLEC4A or variant coding sequences) using LipofectAMINE Plus Reagent (Life Technologies) for 24 h. Later, retroviruses were collected from the culture supernatant and concentrated by centrifugation (8,000 g, 16 hours, 4°C).

The obtained retrovirus was transfected with DOTAP liposomal transfection reagent (DOTAP The cells were infected by treatment with Liposomal Transfection Reagent (Roche) for 2 days. From the obtained cells, a transgenic cell line was purified and separated using a FACSAria ^™ II cell sorter (BD Biosciences) using GFP expression as an indicator.

Expression of GFP and CLEC4A in isolated transgenic cell lines (CLEC4A-expressing cell lines or CLEC4A mutant-expressing cell lines, control retrovirus-infected cell lines) and mouse dendritic cell line DC2.4 was measured by flow cytometry as described above. It was analyzed using the method.

GFP and CLEC4A in mouse dendritic cell line DC2.4 (DC2.4 in the figure), control retrovirus-infected cell line (mock-GFP in the figure), and CLEC4A-expressing cell line (CLEC4A-GFP in the figure) The expression of is shown as a dot plot in FIG. 10A.

Cell line expressing a glycosylation site substitution mutant (in the figure, CLEC4A _N185Q -GFP), cell line expressing a CRD-EPS deletion mutant (in the figure, CLEC4A _ΔE195-S197 -GFP), cell line expressing an extracellular region deletion mutant (In the figure, CLEC4A _ΔF69-L237 -GFP) and the ITIM sequence deletion mutant-expressing cell line (In the figure, CLEC4A _ΔI5-V10 -GFP) The expression of GFP and CLEC4A is shown as a dot plot in FIG. 10B.

Mouse dendritic cell line DC2.4, control retrovirus-infected cell line, CLEC4A-expressing cell line, glycosylation site substitution mutant-expressing cell line, CRD-EPS-deficient mutant-expressing cell line, extracellular domain-deficient mutant-expressing cell line strain, and a cell line expressing an ITIM sequence deletion mutant were respectively seeded in 48-well culture plates (BD Bioscience) and treated with lipopolysaccharide (LPS; 0.1 μg/ml; Sigma-Aldrich), a TLR4 ligand, or CpG, a TLR9 ligand. -B ODN 1668 (0.1 μM) was added for stimulation and cultured for 16 hours, followed by IL-6 (interferon-6; eBioscience) and TNF-α (tumor necrosis factor-α; eBioscience) in the culture supernatant. The amount of was measured by ELISA method. As a control, the same cell line was cultured for 16 hours without stimulation without the addition of lipopolysaccharide or CpG-B ODN 1668, and the amounts of IL-6 and TNF-α in the culture supernatant were similarly measured by ELISA method. . The results are shown in FIG.

Compared to the control retrovirus-infected cell line (mock-GFP in the figure), the CLEC4A-expressing cell line (CLEC4A-GFP in the figure) was stimulated with LPS or CpG-B ODN 1668 to increase IL-6 and TNF- The production of α was markedly attenuated (FIG. 11).

Compared to a CLEC4A-expressing cell line (CLEC4A-GFP in the figure), a cell line expressing a CRD-EPS-deficient mutant (CLEC4A _ΔE195-S197 -GFP in the figure) and a cell line expressing an extracellular domain-deficient mutant (in the figure , CLEC4A _ΔF69-L237 -GFP) and ITIM sequence-deficient mutant-expressing cell lines (CLEC4A _ΔI5-V10 -GFP in the figure), stimulation with LPS or CpG-B ODN 1668 inhibits the production of IL-6 and TNF-α. was significantly enhanced, and the production amount was almost equivalent to that of the control retrovirus-infected cell line (mock-GFP in the figure) (Figure 11). It was shown that CLEC4A mutants introduced into these cell lines do not retain CLEC4A activity.

On the other hand, in the cell line expressing the glycosylation site substitution mutant (CLEC4A _N185Q -GFP in the figure), compared to the CLEC4A expressing cell line, stimulation with LPS or CpG-B ODN 1668 increases IL-6 and TNF-α. Partial enhancement of the production of was observed (FIG. 11). This sugar chain modification site substitution mutant was thought to partially retain CLEC4A activity. N-linked sugar chain (N-type modified sugar chain) at position 185 (N-186) in CRD is required for self-molecule (intra/inter) binding of Clec4A4.

From the above results, CLEC4A is a functional regulatory molecule of antigen-presenting cells, and the molecular basis for the functional regulation of CLEC4A in antigen-presenting cells such as dendritic cells is that the carbohydrate recognition domain (CRD) in its extracellular region and the N An ITIM-dependent inhibitory signal pathway based on self-molecule (intra/inter) binding through association with type-modified sugar chains is involved, and induction of this pathway suppresses cytokine production ability and T cell activation ability. Rukoto has been shown. In other words, it was shown that the extracellular region, CRD, and ITIM sequence are important for the functional regulatory activity of CLEC4A.

[Example 4]
1) Preparation of soluble CLEC4A-mouse IgFc chimera molecule
A BamHI recognition sequence (5'-ggatcc -3'), cDNA with an EcoRV recognition sequence (5'-gatatc-3') added to the 3' end was synthesized using GeneArt ^(R) (Life Technologies). Next, the obtained cDNA was treated with restriction enzymes BamHI and A soluble CLEC4A-mouse IgFc chimeric molecule expression vector was created. A soluble CLEC4A- ^mouse IgFc chimera molecule expression vector was transfected into FreeStyle ^TM 293-F cells (Life Technologies) using 293fectin ^TM Transfection Reagent (Life Technologies). Subsequently, the gene-transfected cells were cultured, and soluble CLEC4A-mouse IgFc chimera molecules were purified from the culture supernatant using HiTrap ^™ Protein G HP (GE Healthcare Life Sciences).

2) Preparation of anti-CLEC4A antibody Using the soluble CLEC4A-mouse IgFc chimera molecule prepared above (12.5 μg/1 mouse) and TiterMax ^(R) Gold Adjuvant (27.5 μl/1 mouse; Sigma-Aldrich), The footpads of the mice were immunized twice at 2-week intervals. Furthermore, on the 31st day after the first immunization, a soluble CLEC4A-mouse IgFc chimera molecule (25 μg/mouse) was administered into the tail vein of the mouse. Lymph nodes of the mice were collected 34 days after the first immunization, and the obtained lymph node cells and the P3U1 myeloma cell line were fused using polyethylene glycol 4000 (Wako) to create a large number of hybridomas.

3) Antibody screening Regarding the culture supernatant of the prepared hybridoma, using the reactivity against CLEC4A-expressing cell lines as an indicator, we used the secondary antibody R-phycoerythrin-labeled F(ab') ₂ fragment goat anti-mouse IgG (H+ L) Antibody clones were screened by the flow cytometry method described above using the antibody (Jackson ImmunoResearch Laboratories).

Specifically, the culture supernatant of the prepared hybridoma was added to a mixture of mouse dendritic cell line DC2.4 and the CLEC4A-expressing cell line (CLEC4A-GFP) prepared in Example 3 (mixing ratio 1:1). Then, the reactivity between the antibody and cells was examined using the flow cytometry method described above. Note that by using a mixed cell system, it is possible to analyze the reactivity to each of the mouse dendritic cell line DC2.4 (parent strain) and the CLEC4A-expressing cell line at the same time.

Similarly, control mouse IgG antibody or anti-CLEC4A antibody (clone 9E8) was applied to a mixture of mouse dendritic cell line DC2.4 and CLEC4A-expressing cell line (CLEC4A-GFP) prepared in Example 3 (mixing ratio 1:1). ), and the reactivity between the antibody and cells was examined using the flow cytometry method described above.

In this way, several dozen clones that showed a high level of response specific to CLEC4A-expressing cell lines were established. Mouse anti-CLEC4A antibodies were purified from the culture supernatants of hybridomas selected as highly reactive clones using HiTrap ^™ Protein G HP.

4) Immune checkpoint inhibitory effect of antibodies Peripheral blood monocyte-derived dendritic cells (1x10 ⁵ cells) obtained in Example 2 were seeded in a 96-well culture plate, and control mouse IgG antibody (cont. Ig) (10 μg/ml; Sigma-Aldrich) or the mouse anti-human CLEC4A antibody obtained above (10 μg/ml) after stimulation with LPS (0.1 ng/ml) and culture for 16 hours. The amount of IL-6 in the culture supernatant was measured by ELISA method.

As a result, in the presence of mouse anti-CLEC4A antibodies #1, #2, #3, and #4, the production of the inflammatory cytokine IL-6 by LPS stimulation of peripheral blood monocyte-derived dendritic cells was inhibited by anti-CLEC4A antibodies. It was enhanced approximately 1.5 times compared to the absence of and the presence of control mouse IgG antibody. It was thought that these anti-CLEC4A antibodies acted as CLEC4A function-inhibiting antibodies and controlled (inhibited) the cytokine production suppressing function of CLEC4A in dendritic cells. In other words, these antibodies were shown to have immune checkpoint inhibitory effects.

Human CLEC4A-expressing mouse dendritic cell line DC2.4 (4x10 ⁵ cells) was incubated in the presence or absence of control mouse IgG antibody (100 μg/ml) or mouse anti-human CLEC4A antibody (100 μg/ml) obtained above. After culturing in the presence of the cells for 60 minutes, a soluble human CLEC4A-mouse IgFc chimera molecule (10 μg/ml) was added to react. Thereafter, using the reactivity (binding) of the soluble human CLEC4A-mouse IgFc chimera molecule to the CLEC4A-expressing mouse dendritic cell line DC2.4 as an indicator, a secondary antibody, R-phycoerythrin-labeled F(ab') ₂ The binding inhibition rate was analyzed by flow cytometry using fragmented goat anti-mouse IgG (H+L). Mouse anti-CLEC4A antibodies #1, #2, and #3 inhibited the binding of soluble CLEC4A-mouse IgFc chimeric molecules to the CLEC4A-expressing mouse dendritic cell line DC2.4 by more than 80%. It was shown that these anti-CLEC4A antibodies inhibited the binding between CLEC4A expressed on the cell surface of the dendritic cell line DC2.4 and soluble CLEC4A.

[Example 5]
Epitope mapping of anti-CLEC4A antibodies #1, #2, #3, and #4, which are CLEC4A function-inhibiting antibodies, was performed using the PEPperMAP ^(R) (PEPperPRINT) peptide microarray contract analysis service.

Specifically, in epitope mapping, a group of overlapping peptides of 15 amino acids in length (overlapping length : 14 amino acids; 155 peptides) were printed on the microarray. Furthermore, anti-CLEC4A antibodies #1, #2, #3, and #4 and labeled secondary antibodies were used to hybridize with the peptide probes on the microarray by antigen-antibody reaction. After scanning the microarray after hybridization using a microarray scanner, reactivity was analyzed using PepSlide ^(R) Analyzer (PEPperPRINT) and epitopes were identified. The consensus motif was an amino acid sequence commonly contained in consecutively overlapping peptide probes that showed strong binding to the anti-CLEC4A antibody. A consensus motif indicates the presence of a linear (contiguous) epitope or a portion of a linear (contiguous) epitope contained within a discontinuous epitope.

The analysis results of each anti-CLEC4A antibody clone were as follows.

- Antibody #1
Epitope mapping identified WVDQTPYNESSTFW (SEQ ID NO: 15; W178-W191 of SEQ ID NO: 2) as a consensus motif. The epitope of antibody #1 was shown to be constituted by a peptide sequence on the CLEC4A protein that includes the consensus motif WVDQTPYNESSTFW (SEQ ID NO: 15).

- Antibody #2
Epitope mapping identified QNLQEESAYF (SEQ ID NO: 16; Q155-F164 of SEQ ID NO: 2) as a consensus motif. Binding of antibody #2 to the peptide FSSNCYFISTESASW (SEQ ID NO: 17; F113-W127 of SEQ ID NO: 2) was also demonstrated. The epitope of antibody #2 was shown to be constituted by a peptide sequence containing the consensus motif QNLQEESAYF (SEQ ID NO: 16) and FSSNCYFISTESASW (SEQ ID NO: 17) on the CLEC4A protein. Antibody #2 can bind to two peptide sequences, QNLQEESAYF (SEQ ID NO: 16) and FSSNCYFISTESASW (SEQ ID NO: 17), which are located far apart on the primary sequence of CLEC4A protein. 16) and at least some of the amino acids in FSSNCYFISTESASW (SEQ ID NO: 17).

- Antibody #3
Epitope mapping identified SSNCYFISTESASW (SEQ ID NO: 18; S114-W127 of SEQ ID NO: 2) and QNLQEESAYF (SEQ ID NO: 16; Q155-F164 of SEQ ID NO: 2) as consensus motifs. The epitope of antibody #3 was shown to be constituted by a peptide sequence containing the consensus motif SSNCYFISTESASW (SEQ ID NO: 18) and QNLQEESAYF on the CLEC4A protein. Antibody #3 can bind to two peptide sequences, SSNCYFISTESASW (SEQ ID NO: 18) and QNLQEESAYF (SEQ ID NO: 16), which are located at distant positions on the primary sequence of CLEC4A protein. ) and QNLQEESAYF (SEQ ID NO: 16).

- Antibody #4
Epitope mapping identified KKNMPVEETAWSCC (SEQ ID NO: 19; K93-C106 of SEQ ID NO: 2) as a consensus motif. The epitope of antibody #4 was shown to be constituted by a peptide sequence on the CLEC4A protein that includes the consensus motif KKNMPVEETAWSCC (SEQ ID NO: 19).

FIG. 12 shows the positions of the antibody binding sequences constituting the epitopes of antibodies #1 to #4 on the primary sequence of the CLEC4A protein.

It was revealed that the epitopes of antibodies #1 to #4, which are CLEC4A function-inhibiting antibodies, exist within the C-type lectin-like domain of the CLEC4A extracellular region. Furthermore, it was shown that the epitope of antibody #1 includes the N-type glycosylation site (N185) within the CRD (FIG. 12).

[Example 6]
1) Preparation of CLEC4A transgenic mouse The 5' end and 3 of the nucleotide sequence (SEQ ID NO: 1) encoding the full-length human CLEC4A protein (SEQ ID NO: 2; NCBI accession number NP_057268.1; M1-L237; 237 amino acid length) A cDNA (SEQ ID NO: 20) with a ClaI recognition sequence (5'-atcgat-3') added to the 'end was synthesized using GeneArt ^(R) (Life Technologies).

To generate human CLEC4A transgenic mice, we used the pDOI-6 vector (van Santen H ., et al., J. Immunol. Methods, (2000) 245: 133-137).

After treating the above synthetic cDNA with the restriction enzyme ClaI, it was introduced into the ClaI site in the multi-cloning site of the pDOI-6 vector to produce a CLEC4A expression pDOI-6 vector. The CLEC4A expression pDOI-6 vector was linearized by treatment with restriction enzymes XhoI and PvuI and purified. The linearized CLEC4A-expressing pDOI-6 vector was microinjected into the pronucleus of a fertilized egg of mouse C57BL/6 strain (CLEA Japan, Inc.), and then the fertilized egg was transplanted into the fallopian tube of a pseudopregnant mouse. After weaning, the tails of the offspring obtained after pregnancy and birth were collected, and genomic DNA was extracted. Genotyping PCR analysis was performed using the obtained genomic DNA, and a founder mouse (F0) into which CLEC4A was introduced was identified. Furthermore, F1 mice were generated by crossing the founder mouse (F0) with C57BL/6 mice, and human CLEC4A transgenic mice were established by genotyping PCR analysis. The genetic recombination experiments and animal experiments conducted were approved by the affiliated institution.

2) CLEC4A expression in immune cells of human CLEC4A transgenic mice Red blood cell lysis buffer (Red Splenic mononuclear cells were prepared using Blood Cell Lysing Buffer Hybri-Max ^™ (Sigma-Aldrich) (Non-Patent Document 4). The expression of CLEC4A in CD3 ⁺ T cells, CD19 ⁺ B cells, CD11b ⁺ macrophages, CD11c ⁺ dendritic cells, and NK1.1 ⁺ NK cells in splenic mononuclear cells was analyzed by the flow cytometry method described above. FIG. 13A shows a histogram of the expression of CLEC4A in cells positive for expressing each cell surface molecule (CD3, CD19, CD11b, CD11c, and NK1.1). Numbers indicate average fluorescence intensity.

No expression of CLEC4A was observed in splenic mononuclear cells of wild-type mice (WT). On the other hand, CLEC4A expression was observed in splenic mononuclear cells of CLEC4A-expressing transgenic mice (CLEC4A Tg), and especially CD11c ⁺ dendritic cells showed higher CLEC4A expression levels compared to other immune cells (Fig. 13A).

Additionally, splenic dendritic cell subpopulations (CD11c ⁺ CD8α ⁺ cDC1 cells, CD11c ⁺ CD4 ⁺ cDC2 cells) in splenic mononuclear cells from wild-type mice (WT; C57BL/6) and CLEC4A-expressing transgenic mice (CLEC4A Tg) , and CD11c ⁺ SiglecH ⁺ pDC cells) was analyzed by the flow cytometry method described above. FIG. 13B shows a histogram of CLEC4A expression in each splenic dendritic cell subpopulation. Numbers indicate average fluorescence intensity.

CLEC4A expression was not observed in any of the splenic dendritic cell subpopulations of wild-type mice (WT). On the other hand, CLEC4A expression was observed in the splenic dendritic cell subpopulations of CLEC4A-expressing transgenic mice (CLEC4A Tg), with higher CLEC4A in CD11c ⁺ CD8α ⁺ cDC1 cells, CD11c ⁺ SiglecH ⁺ pDC cells, and CD11c ⁺ CD4 ⁺ cDC2 cells. Expression levels are shown (Figure 13B).

It was confirmed that the created human CLEC4A transgenic mouse expressed human CLEC4A in immune cells.

[Example 7]
1) Effect of suppressing cancer progression in tumor-bearing mice by CLEC4A function-inhibiting antibody Ovalbumin ( By subcutaneously transplanting (1x10 ⁵ cells/1 animal) a recombinant malignant melanoma cell line (B16-OVA melanoma; Brown DM, et al., Immunology. (2001) 102:486-497) expressing OVA). , we generated tumor-bearing mice.

After the tumor volume of the tumor-bearing CLEC4A-expressing transgenic mouse reached approximately 100 ^m3 (6 days after cancer cell transplantation), control mouse IgG antibody (cont. Ig (Sigma-Aldrich); 100 μg/mouse) or CLEC4A Function-inhibiting antibodies (antibodies #1 to #4; 100 μg/mouse) were administered intraperitoneally a total of 6 times at 3-day intervals. Cancer progression was evaluated using a digital caliper (Digimatic Caliper, Mitutoyo) to measure the tumor volume of tumor-bearing mice every day until 22 days after cancer cell transplantation.

In addition, as indicators of immune-related adverse events, changes in body weight, poor food and water intake, and external changes (immobility, piloerection, etc.) of the tumor-bearing mice were observed up to 22 days after tumor transplantation.

FIG. 14A shows a photograph of the tumor 13 days after cancer cell transplantation. Figures 14B and C show tumor volumes 13 days (B) and 22 days (C) after cancer cell transplantation. Figure 15 shows changes in tumor volume over time for 22 days after cancer cell transplantation.

In tumor-bearing CLEC4A-expressing transgenic mice, administration of CLEC4A function-inhibiting antibodies (antibodies #1 to #4) significantly inhibited cancer progression compared to administration of control antibodies (cont. Ig). A remarkable therapeutic effect on cancer was observed (Figures 14 and 15).

Furthermore, in the CLEC4A function-inhibiting antibody administration group, no weight loss was observed compared to the control group (Figure 16), and no poor water intake or changes in appearance (immobilization, piloerection, etc.) were observed. In other words, it was shown that the CLEC4A function-inhibiting antibody did not cause immune-related adverse events (irAEs).

2) Effect of promoting tumor-infiltrating T cell accumulation by CLEC4A function-inhibiting antibody Tumor-bearing mice were prepared in the same manner as in 1) above, and after the tumor volume reached approximately 100 m ³ , a control mouse IgG antibody (cont. Ig; 100 μg /1 animal) or CLEC4A function-inhibiting antibody (antibodies #1 to #4; 100 μg/1 animal) was administered intravenously a total of 6 times at 3-day intervals. Cancer tissues were collected 22 days after cancer cell transplantation. The accumulation of tumor-infiltrating CD4 ⁺ T cells and tumor-infiltrating CD8 ⁺ T cells in cancer tissues was analyzed using the flow cytometry method described above.

The results of flow cytometry analysis are shown in FIG. 17. In the CLEC4A function-inhibiting antibody administration group, a significant accumulation of tumor-infiltrating CD4 ⁺ T cells and tumor-infiltrating CD8 ⁺ T cells was observed in cancer tissues compared to the control antibody administration group. It was shown that an antibody that inhibits CLEC4A function promotes the accumulation of tumor-infiltrating T cells into cancer tissues.

When the results shown in Example 7 are considered together with the results of Examples 1 to 6, it is clear that CLEC4A function-inhibiting antibodies #1 to #4 have a peptide sequence within the C-type lectin-like domain present in the extracellular region of CLEC4A. By binding to CLEC4A-expressing dendritic cells, it inhibits the ITIM-dependent inhibitory signal pathway based on self-molecule binding through the association of CRD and N-type modified sugar chains, and enhances the function of dendritic cells, especially CLEC4A-expressing dendritic cells. This study shows that by enhancing the antigen-presenting function of cancer cells, it amplifies the induction of cancer antigen-specific cytotoxic T cells (CTLs) and suppresses cancer progression.

Furthermore, since CLEC4A function-inhibiting antibodies #1 to #3 had particularly high cancer progression suppressing effects, antibody binding to the F113 to H192 region (SEQ ID NO: 21) of the human CLEC4A protein to which these antibodies bind It was shown to result in higher antitumor activity.

Antibodies #1 to #4 also do not cause immune-related adverse events (irAEs), so they are considered to be safer than existing immune checkpoint inhibitors for CTLA-4/PD-1. it was thought.

CLEC4A is an "immune checkpoint molecule" that controls cancer immune (autoimmune) responses, and antibodies #1 to #4, which have immune checkpoint inhibitory effects, can be used as immune checkpoint inhibitors for cancer immunotherapy. It is promising for applications. CLEC4A function-inhibiting antibodies #1 to #4 cause immune checkpoint inhibition through a different mechanism of action from existing immune checkpoint inhibitors against the CTLA-4/PD-1 system, so they are not compatible with existing immune checkpoint inhibitors. Effective anti-tumor effects can be expected even against cancer types that exhibit non-responsiveness or acquired resistance.

[Example 8]
1) Effect of suppressing cancer progression of CLEC4A function-inhibiting antibodies and anti-PD-1 neutralizing antibodies in tumor-bearing mice Wild-type mice (WT; C57BL/6) and CLEC4A-expressing transgenic mice established in Example 6 (CLEC4A Tg A recombinant malignant melanoma cell line (B16-OVA melanoma; Brown DM, et al., Immunology. (2001) 102:486-497) expressing ovalbumin (OVA) was subcutaneously implanted (1x10 ⁵ Tumor-bearing mice (tumor-bearing CLEC4A-expressing transgenic mice) were generated by cell/1 mouse).

After the tumor volume of tumor-bearing CLEC4A-expressing transgenic mice reached approximately 100 ^m3 (8 days after cancer cell transplantation), (i) control mouse IgG antibody (cont. Ig (Sigma-Aldrich); 100 μg/mouse ), (ii) CLEC4A function-inhibiting antibody (antibody #1; 100 μg/1 animal), (iii) anti-PD-1 neutralizing antibody (clone 29F.1A12 (Biolegend); 200 μg/1 animal), or (iv) CLEC4A A combination of a function-blocking antibody (antibody #1; 100 μg/mouse) and an anti-PD-1 neutralizing antibody (clone 29F.1A12 (Biolegend); 200 μg/mouse) was administered intraperitoneally 5 times at 3-day intervals. did. As a control, an untreated group in which nothing was administered to tumor-bearing mice was also prepared. Cancer progression was evaluated using a digital caliper (Digimatic Caliper, Mitutoyo) to measure the tumor volume of tumor-bearing mice every day until 21 days after cancer cell transplantation.

21 days after cancer cell transplantation, the tumor-bearing mice were euthanized after cancer progression was evaluated, and skin tissue, lung tissue, liver tissue, and small intestine tissue were collected from the tumor-bearing mice and treated with 4% paraformaldehyde. -Fixed in phosphate buffered saline and embedded in paraffin. Furthermore, after slicing these paraffin-embedded tissues, tissue sections (5 μm thick) were prepared and stained with hematoxylin and eosin.

The results of tissue staining are shown in Figure 18. In tumor-bearing CLEC4A-expressing transgenic mice, administration of the CLEC4A function-blocking antibody (antibody #1) did not cause tissue damage in the skin, lungs, liver, or small intestine, even when compared with untreated mice or administration of a control mouse IgG antibody (cont. Ig) (Figure 18). As shown in Example 7, the CLEC4A function-blocking antibody does not cause immune-related adverse events (irAEs), such as weight loss, and does not cause immune-related adverse events (irAEs) indicated by tissue damage, demonstrating its high safety.

Figure 19 shows the results of cancer progression evaluation. FIG. 19A shows the time course of tumor volume in tumor-bearing mice 21 days after cancer cell transplantation. FIG. 19B shows the tumor volume in the tumor-bearing mouse 21 days after cancer cell transplantation, and FIG. 19C shows a photograph of the tumor-bearing mouse and the excised tumor 21 days after cancer cell transplantation. Compared to untreated or administration of control mouse IgG antibody (cont. Compared to single administration of the Japanese antibody (clone 29F.1A12), the effect of inhibiting cancer progression was even higher (FIGS. 19A to 19C). Furthermore, the combined administration of CLEC4A function-inhibiting antibody (antibody #1) and anti-PD-1 neutralizing antibody (clone 29F.1A12) was more effective than the single administration of CLEC4A function-inhibiting antibody (antibody #1). It showed a strong cancer progression inhibiting effect (FIGS. 19A to 19C). The combination of a CLEC4A function-inhibiting antibody and an anti-PD-1 neutralizing antibody against the immune checkpoint molecule PD-1 expressed in T cells was shown to have a more powerful cancer therapeutic effect. These results showed that the combination of a CLEC4A function-blocking antibody and an inhibitor against immune checkpoint molecules expressed in T cells produces a synergistic cancer therapeutic effect.

2) Effect of CLEC4A function-inhibiting antibody and anti-PD-1 neutralizing antibody on cancer tissue-infiltrating immune cells and cancer microenvironment control in tumor-bearing mice CLEC4A-expressing transgenic mouse (CLEC4A Tg) established in Example 6 A recombinant malignant melanoma cell line (B16-OVA melanoma; Brown DM, et al., Immunology. (2001) 102:486-497) expressing ovalbumin (OVA) was subcutaneously implanted (1x10 ⁵ cells) on the back of the patient. /1 mouse) to generate tumor-bearing mice (tumor-bearing CLEC4A-expressing transgenic mice).

After the tumor volume of tumor-bearing CLEC4A-expressing transgenic mice reached approximately 100 ^m3 (8 days after cancer cell transplantation), (i) control mouse IgG antibody (cont. Ig (Sigma-Aldrich); 100 μg/mouse ), (ii) CLEC4A function-inhibiting antibody (antibody #1; 100 μg/1 animal), (iii) anti-PD-1 neutralizing antibody (clone 29F.1A12 (Biolegend); 200 μg/1 animal), or (iv) CLEC4A A combination of a function-blocking antibody (antibody #1; 100 μg/mouse) and an anti-PD-1 neutralizing antibody (clone 29F.1A12 (Biolegend); 200 μg/mouse) was administered intraperitoneally 5 times at 3-day intervals. did. As a control, an untreated group in which nothing was administered to tumor-bearing mice was also prepared.

Cancer tissue was collected from a tumor excised 21 days after cancer cell transplantation from a tumor-bearing CLEC4A-expressing transgenic mouse that underwent the cancer progression evaluation test in 1) above, and cells within the tissue were obtained.

In order to investigate immune cell dynamics in cancer tissues, the percentage of each immune cell in cancer-infiltrating leukocytes was analyzed using flow cytometry. Figure 20A shows the expression of each cell surface molecule as a dot plot.

In tumor-bearing CLEC4A-expressing transgenic mice, administration of CLEC4A function-inhibiting antibody (antibody #1) alone or anti-PD-1 neutralizing antibody (clone 29F.1A12) compared to untreated or administration of control mouse IgG. It was shown that single administration suppressed the accumulation of Gr1-1 ⁺ CD11b ⁺ F4/80 ⁺ myeloid-derived suppressor cells (MDSCs) in cancer tissues (FIG. 20A). In addition, single administration of CLEC4A function-inhibiting antibody (antibody #1) or anti-PD-1 neutralizing antibody (clone 29F.1A12) showed that CD11c ⁺ SiglecH ^- cDCs (conventional dendritic cells), CD11c ^low SiglecH ⁺ Increased accumulation of pDCs (plasmacytoid dendritic cells), NK1.1 ⁺ NK cells, and cancer tissue (tumor)-infiltrating CD4 ⁺ TILs/CD8 ⁺ TILs in cancer tissues was observed (FIG. 20A).

Furthermore, to examine the expression status of immunosuppressive molecules in cancer tissues, the expression of each immunosuppressive molecule in cancer tissues 21 days after cancer cell transplantation was analyzed using quantitative PCR (RT-PCR). Total RNA was prepared from cells obtained from cancer tissues using the RNeasy plus micro kit (Qiagen), and then the total RNA was used as a template and oligo(dT)20 primers using the PrimeScript RT reagent kit (Takara). cDNA was synthesized. Quantitative PCR was performed using Thermal Cycler Dice (Takara) using the following primer sets specific to each immunosuppressive molecule and SYBR ^(R) Premix Ex Taq II (Takara). The expression level of each immunosuppressive molecule obtained by quantitative PCR was normalized to the expression level of the reference gene glyceraldehyde phosphate dehydrogenase gene (gapdh).

a) IL-10 gene specific primer set (il10):
5'-TGCAGCAGCTCAGAGGGTT-3' (SEQ ID NO: 33)
5'-TGGCCACAGTTTTCAGGGAT-3' (SEQ ID NO: 34)
b) TGF-β gene specific primer set (tgfb):
5'-ACCATGCCAACTTCTGTCTG-3' (SEQ ID NO: 35)
5'-CGGGTTGTGTTGGTTGTAGA-3' (SEQ ID NO: 36)
c) IDO gene-specific primer set (ido):
5'-TCGGAAGAGCCCTCAAATGTGGAA-3' (SEQ ID NO: 37)
5'-TGGAGCTTGCTACACTAAAGGCCAA-3' (SEQ ID NO: 38)
d) Arginase gene-specific primer set (ariginase):
5'-AAGACAGCAGAGGAGGTGAAG-3' (SEQ ID NO: 39)
5'-TAGTCAGTCCCTGGCTTATGG-3' (SEQ ID NO: 40)
e) iNOS gene specific primer set (inos):
5'-AACAATTCCTGGCGTTACCTT-3' (SEQ ID NO: 41)
5'-TGTATTCCGTCTCCTTGGTTC-3' (SEQ ID NO: 42)
f) COX-2 gene specific primer set (cox2):
5'-TGGGTGTGAAGGGAAATAAGG-3' (SEQ ID NO: 43)
5'-CATCATATTTGAGCCTTGGGG-3' (SEQ ID NO: 44)
g) mPGES gene specific primer set (mpges):
5'-AGGATGCGCTGAAAACGTGGAG-3' (SEQ ID NO: 45)
5'-CCGAGGAAGAGGAAAGGATAG-3' (SEQ ID NO: 46)
h) GAPDH gene specific primer set (gapdh):
5'-AAATTCAACGGCACAGTCAAG-3' (SEQ ID NO: 47)
5'-TGGTGGTGAAGACACCAGTAG-3' (SEQ ID NO: 48)

The analysis results are shown in FIG. 20B. In tumor-bearing CLEC4A-expressing transgenic mice, single administration of the CLEC4A function-inhibiting antibody (antibody #1) increased IL, an immunosuppressive molecule, in cancer tissue compared to untreated or administration of control mouse IgG. (interleukin)-10, IDO (indoleamine 2,3-dioxygenase), arginase, and mPGES (membrane-associated prostaglandin E2 synthase) expression was suppressed. In addition, single administration of anti-PD-1 neutralizing antibody (clone 29F.1A12) was more effective than administration of untreated or control mouse IgG. )-2, and mPGES expression was suppressed. Furthermore, the combined administration of the CLEC4A function-inhibiting antibody (antibody #1) and the anti-PD-1 neutralizing antibody (clone 29F.1A12) is effective against TGF (transforming growth factor)-β and iNOS (inducible nitric oxide synthase). ), and showed a stronger inhibitory effect on the expression of IL-10, IDO, arginase, COX-2, and mPGES than when each was administered alone.

From the above results, we found that CLEC4A function-inhibiting antibodies suppress the expression of immunosuppressive molecules in cancer tissues, and that CLEC4A function-inhibiting antibodies and anti-PD, an inhibitor of PD-1, an immune checkpoint molecule expressed in T cells. The combination of -1 neutralizing antibody was shown to suppress the expression of immunosuppressive molecules more strongly.

Administration of CLEC4A function-inhibiting antibodies and anti-PD-1 neutralizing antibodies has the effect of controlling the cancer microenvironment, and in particular suppresses the expression of immunosuppressive molecules in cancer tissues and suppresses immune tolerogenicity in the cancer microenvironment. It was thought that by suppressing and inducing immunogenicity of cancer cells (tumors), it would improve the cancer microenvironment and enhance the cancer immune response. Furthermore, the combination of CLEC4A function-blocking antibodies and immune checkpoint inhibitors, such as inhibitors of immune checkpoint molecules expressed on T cells, inhibits the function of immune checkpoint molecules in different immune cell lineages and disrupts the cancer microenvironment. It can be said that a synergistic cancer therapeutic effect can be brought about by strongly improving (inducing) immunogenicity from tolerogenicity.

The present invention provides a novel cancer therapy that can effectively induce immune checkpoint inhibition through functional inhibition of the immune checkpoint molecule CLEC4A, activate antitumor immune responses, and is highly safe. It can be used as a therapeutic tool.

All publications, patents, and patent applications cited herein are incorporated by reference in their entirety.

SEQ ID NO: 1: Full-length CLEC4A coding sequence SEQ ID NO: 2: Full-length CLEC4A protein SEQ ID NO: 3: ITIM (I5-V10) deletion mutant coding sequence SEQ ID NO: 4: ITIM (I5-V10) deletion mutant SEQ ID NO: 5: Extracellular region (F69-L237) deletion mutant coding sequence SEQ ID NO: 6: Extracellular region (F69-L237) deletion mutant SEQ ID NO: 7: CRD-EPS (E195-S197) deletion mutant coding sequence SEQ ID NO: 8: CRD-EPS( E195-S197) deletion mutant SEQ ID NO: 9: N185Q variant coding sequence SEQ ID NO: 10: N185Q variant SEQ ID NO: 11: BamHI and XhoI site addition sequence SEQ ID NO: 12: CLEC4A extracellular region (F69-L237)
Sequence number 13: CLEC4A ITIM sequence (I5-V10)
SEQ ID NO: 14: OVA257-264 peptide SEQ ID NO: 15: Antibody #1 binding sequence SEQ ID NO: 16: Antibody #2 and #3 binding sequence SEQ ID NO: 17: Antibody #2 binding sequence SEQ ID NO: 18: Antibody #3 binding sequence SEQ ID NO: 19 : Antibody #4 binding sequence SEQ ID NO: 20: ClaI site addition fragment SEQ ID NO: 21: CLEC4A F113-H192
SEQ ID NO: 22-32: Synthetic peptide SEQ ID NO: 33-48: Primer

Claims (12)

An antitumor drug for activating an antitumor immune response based on immune checkpoint inhibition, which specifically binds to the extracellular region of human CLEC4A protein and has an immune checkpoint inhibitory effect or its antigen binding Contains sexual fragments,
The antibody or antigen-binding fragment thereof:
(i) specifically binds to the region consisting of the amino acid sequence shown by SEQ ID NO: 21 in the extracellular region of human CLEC4A protein, or (ii) specifically binds to the peptide fragment consisting of the amino acid sequence KKNMPVEETAWSCC (SEQ ID NO: 19) join to,
Antitumor drug. The antitumor drug according to claim 1, wherein the antibody or antigen-binding fragment thereof specifically binds to a region consisting of the amino acid sequence shown by SEQ ID NO: 21 in the extracellular region of human CLEC4A protein. The antitumor drug according to claim 1 or 2, which specifically binds to a peptide fragment consisting of the amino acid sequence WVDQTPYNESSTFW (SEQ ID NO: 15). Claims 1 to 3, wherein the antibody or antigen-binding fragment thereof is capable of binding to a peptide fragment consisting of an amino acid sequence with a length of 15 amino acids including WVDQTPYNESSTFW (SEQ ID NO: 15) on the amino acid sequence shown by SEQ ID NO: 2. The antitumor drug according to any one of the above. The antitumor drug according to claim 1, wherein the antibody or antigen-binding fragment thereof specifically binds to a peptide fragment consisting of the amino acid sequence KKNMPVEETAWSCC (SEQ ID NO: 19). Claim 1 or 5, wherein the antibody or antigen-binding fragment thereof is capable of binding to a peptide fragment consisting of a 15 amino acid long amino acid sequence including KKNMPVEETAWSCC (SEQ ID NO: 19) on the amino acid sequence shown by SEQ ID NO: 2. The antitumor drug described in . The antitumor drug according to any one of claims 1 to 6 , for use in combination with an inhibitor against immune checkpoint molecules expressed in T cells. The antitumor drug according to any one of claims 1 to 6 , further comprising an inhibitor against immune checkpoint molecules expressed in T cells. The antitumor drug according to claim 7 or 8 , wherein the immune checkpoint molecule expressed on T cells is PD-1. The antitumor drug according to any one of claims 7 to 9 , wherein the inhibitor against immune checkpoint molecules expressed in T cells is an anti-PD-1 antibody. The antitumor drug according to any one of claims 1 to 10 , which is used to suppress the progression of cancer. The antitumor drug according to any one of claims 1 to 11 , for intravenous or intraperitoneal administration.

Images