Use for preventing and treating diseases related to myeloid-derived suppressor cells

Abstract

The present invention relates to an anti-CD66c antibody that binds to CD66c expressed on MDSC and its use, and to an anti-CD66c antibody that binds to MDSC, and a pharmaceutical composition and a diagnostic composition comprising the same. The anti-CD66c antibody of the present invention targets MDSC that induces immunosuppression, and can therefore be used to treat a variety of diseases caused by MDSC.

Description

Technical Field

[0001] The present invention relates to an immunopotentiator and the use of the immunopotentiator to prevent, improve or treat MDSC-related diseases, wherein the immunopotentiator comprises an antibody or an antigen-binding fragment thereof that specifically binds to CD66c expressed in myeloid-derived suppressor cells (MDSCs). Specifically, the present invention provides a method for reducing the immunosuppressive activity of MDSCs by regulating production, death or activity with a monoclonal antibody, thereby preventing, improving or treating MDSC-related diseases or diagnosing the same. Background Art

[0002] Recently, immunotherapy using antibodies or immune cell vaccines has been actively studied in cancer treatment. However, the immune escape and suppression of cancer cells inhibit the therapeutic effect. For the purpose of preventing the immune response to cancer cells themselves, cancer cells reduce the activity of various immune cells and induce cells with immunosuppressive functions, such as inactivated dendritic cells, regulatory T cells (Treg) and tumor-associated macrophages (TAM). Recently, as one of the immunosuppressive cells, the role of myeloid-derived suppressor cells has received widespread attention.

[0003] MDSCs are defined as a collection of immature myeloid cells of bone marrow origin with immunosuppressive functions. It has been reported that, although the number of MDSCs is limited in healthy individuals, they accumulate in peripheral blood, lymphoid organs, spleen, and cancer tissues under pathological conditions such as chronic / acute infection and cancer.

[0004] MDSCs can also promote the growth of cancer cells and induce distant metastasis of cancer cells by suppressing the immune response of T cells and NK cells and inducing the generation of Treg cells, which are immunosuppressive cells.

[0005] The immunosuppressive mechanisms of MDSC known so far can be divided into four main types. The first is the lack of nutrients required by lymphocytes. The second is the generation of oxidative stress, which inhibits multiple steps from the proliferation to the function of T cells, such as by producing reactive oxygen or reactive nitrogen. The third is to affect the trafficking and survival of lymphocytes. Specifically, mechanisms such as inhibiting the recirculation process of T cells to lymph nodes, preventing T cells from moving to the center of the tumor, and inducing T cell death are known. Fourth, it is known to proliferate antigen-specific natural Treg cells and promote the process of converting naive CD4+T cells into Treg.

[0006] One of the greatest characteristics of MDSC is the diversity of its form, phenotype and function. As markers of MDSC, lineage (-), HLA-DR low / (-), CD11b (+) and CD33 (+) are known. Since these markers are often expressed in several different types of myeloid cells, such as precursor cells of dendritic cells, macrophages and granulocytes, MDSC is defined as a group of myeloid-derived cells with immunosuppressive functions. This diversity of MDSC has led to different analyses in studying the origin and characteristics of MDSC, causing great confusion in the research. Therefore, studies have been conducted to clarify the subpopulations of MDSC, and it is currently found that MDSC consists of 80% granulocyte MDSC and 20% monocyte MDSC. These two cell types differ not only in shape and phenotype, but also in the mechanism of suppressing immunity. Granulocyte MDSC induces antigen-specific immunosuppression by contacting between T cells via reactive oxygen species. Monocyte MDSC mainly exhibits immunosuppressive function by using high expression of arginase and various immunosuppressive cytokines.

[0007] Recent studies have reported that the accumulation of MDSC is associated with the immunosuppressive environment that occurs in cancer patients, which is common in almost all cancer types. Many studies have shown that the degree of MDSC increase increases with the progression of cancer stages. Therefore, studies using the increased degree of MDSC as a prognostic marker for the low survival rate and treatment response rate of cancer patients are actively underway. Obviously, MDSC plays an important role in the pathophysiology of cancer. Summary of the invention

[0008] Technical issues

[0009] An embodiment of the present invention is an immunopotentiator, an immunoactivator or a composition comprising an antibody or an antigen-binding fragment thereof that specifically binds to CD66c expressed in myeloid-derived suppressor cells (MDSCs) and is used to reduce or eliminate the immunosuppressive activity of MDSCs.

[0010] An embodiment of the present invention is a pharmaceutical composition or use for preventing, ameliorating or treating MDSC-related diseases, comprising an antibody or an antigen-binding fragment thereof that specifically binds to CD66c expressed in MDSC.

[0011] An embodiment of the present invention is a method of enhancing or activating an immune response in a subject, the method comprising administering to a subject in need thereof an antibody or antigen-binding fragment thereof that specifically binds to CD66c expressed in MDSCs.

[0012] Another embodiment of the present invention is a method of inhibiting the activity of MDSCs, the method comprising contacting MDSCs with an antibody or an antigen-binding fragment thereof that specifically binds to CD66c expressed in MDSCs.

[0013] In addition, an embodiment of the present invention is a method for preventing, ameliorating or treating an MDSC-related disease, comprising administering an immunopotentiator or an immunoactivator to a subject suffering from an MDSC-related disease.

[0014] In addition, an embodiment of the present invention is a method for preventing, ameliorating or treating MDSC-related diseases, comprising administering an immunopotentiator or an immunoactivator comprising an antibody or an antigen-binding fragment thereof that specifically binds to CD66c expressed in myeloid-derived suppressor cells to a subject suffering from an MDSC-related disease.

[0015] The antibody or antigen-binding fragment thereof that specifically binds to CD66c expressed in myeloid-derived suppressor cells according to the present invention eliminates or reduces the immunosuppressive activity of MDSCs, reduces the number of MDSCs, regulates the activity, generation or cell death of MDSCs, or induces cell death.

[0016] Technical Solution

[0017] The present invention relates to the use of an antibody or an antigen-binding fragment thereof that specifically binds to CD66c expressed in myeloid-derived suppressor cells (MDSCs) for immune enhancement, immune activation, or reduction or elimination of the immunosuppressive activity of MDSCs.

[0018] A further embodiment of the present invention relates to the use of an antibody or an antigen-binding fragment thereof that specifically binds to CD66c expressed in MDSCs for preventing, ameliorating or treating MDSC-related diseases (eg, cancer, infectious diseases, etc.).

[0019] Specifically, the present invention relates to the use of preventing, treating or diagnosing MDSC-related diseases by inducing a decrease in the immunosuppressive activity of MDSC.

[0020] The antibody may be a polyclonal antibody or a monoclonal antibody, and may be a mouse antibody, a chimeric antibody, or a humanized antibody.

[0021] Another embodiment provides a nucleic acid molecule encoding an anti-CD66c antibody or an antigen-binding fragment thereof.

[0022] Another embodiment provides a recombinant vector comprising the nucleic acid molecule. The recombinant vector can be used as an expression vector for expressing the nucleic acid molecule in a host cell.

[0023] A further embodiment provides a recombinant cell comprising a nucleic acid molecule or a recombinant vector. The recombinant cell can be obtained by transforming the nucleic acid molecule or the recombinant vector into a host cell.

[0024] Another embodiment provides a method for preparing an anti-CD66c antibody or an antigen-binding fragment thereof. The preparation method may include the step of expressing a nucleic acid molecule in a host cell. The expression step may include culturing recombinant cells, and optionally, may also include isolating and / or purifying the antibody from the obtained cell culture. The method may include the following steps:

[0025] (a) preparing a recombinant cell transformed with a nucleic acid molecule or a recombinant vector;

[0026] (b) culturing the recombinant cell under conditions and / or for a period of time sufficient to express the nucleic acid molecule; and

[0027] (c) isolating and / or purifying the anti-CD66c antibody or antigen-binding fragment thereof from the culture obtained in step (c).

[0028] Hereinafter, the present invention will be described in more detail.

[0029] In one embodiment, the preparation method relates to a composition, an immunopotentiator or an immunoactivator for reducing or eliminating the immunosuppressive capacity of MDSC, which comprises an antibody or an antigen-binding fragment thereof that binds to CD66c expressed in MDSC.

[0030] MDSCs are defined as a collection of bone marrow-derived immature myeloid cells with immunosuppressive functions, and are reported to accumulate in peripheral blood, lymphoid organs, spleen, cancer tissues, etc. under pathological conditions such as chronic / acute infections and cancer.

[0031] MDSC promotes the growth of cancer cells and can also induce distant metastasis of cancer cells by suppressing the immune response of T cells and NK cells and inducing the production of Treg cells as immunosuppressive cells. The immunosuppressive mechanisms of MDSC known so far are the depletion of nutrients required by lymphocytes, affecting the trafficking and survival of lymphocytes, generating oxidative stress that inhibits multiple steps of T cell proliferation to function by producing reactive oxygen or reactive nitrogen, or blocking the movement of T cells to the center of cancer tissue, and inducing cell death of T cells. In addition, MDSC is known to proliferate antigen-specific natural Treg cells and promote the process of converting naive CD4+T cells into Tregs.

[0032] MDSC is defined as a collection of immature bone marrow cells of bone marrow origin with immunosuppressive function. Although the number is limited in healthy individuals, it accumulates in peripheral blood, lymphoid organs, spleen and cancerous tissues under pathological conditions such as chronic / acute infection and cancer. It has been reported that MDSC accumulation and immunosuppressive function in cancer in colon cancer, fibrosarcoma, thymoma, lung cancer, mesothelioma, lymphoma, prostate cancer, head and neck cancer, melanoma, etc. (Gabrilovich DI et al., Tumors to the coordinated regulation of myeloid cells (Coordinated regulation of myeloid cells by tumors), Nat Rev Immunol.12 (4): 253-68). In addition to cancer, MDSCs are known to induce immunosuppression in diseases such as infection with Trypanosoma cruzi, Listeria monocytogenes, Leishmania major, helminths, Candida albicans, Porphyromonas gingivalis, or toxoplasmosis and polymicrobial sepsis (Garbrilovich DI et al., Myeloid-derived suppressor cells as regulators of the immune systems. Nat Rev Immunol. 9(3): 162-74 (2009)).

[0033] In the present disclosure, MDSCs with a phenotype of non-lymphoid HLA-DR low / (-), CD11b+ and CD33+ and expressing CD66c can be targets of anti-CD66c antibodies or antigen-binding fragments thereof according to the present invention. In particular, the present invention can target the accumulation of CD66c-positive MDSCs in MDSCs with a phenotype of non-lymphoid HLA-DR low / (-), CD11b+ and CD33+, thereby proposing a plan to improve or treat immunodeficiency, decreased immunity, and immune damage caused by MDSCs. For example, by specifying the monocyte area and granulocyte area other than lymphocytes according to cell size in the dot plot, selecting a group in which HLA-DR is not expressed or expressed at a low level, and selecting a group that is CD11b and CD33 positive, MDSCs can be specified.

[0034] Embodiments of the present invention may provide a pharmaceutical composition or use for preventing, ameliorating or treating MDSC-related diseases by using an antibody or an antigen-binding fragment thereof that specifically binds to CD66c expressed in MDSC according to the present invention.

[0035] The lysis effect of the anti-CD66c antibody on MDSC according to the present invention can induce a reduction in the number of MDSC cells or apoptosis of CEACAM6-positive cells in whole blood and PMBC. Preferably, the anti-CD66c antibody can induce a reduction in number or cell death in an ADCC manner. In whole blood, neutrophils that are positive for the CEACAM6 target antigen are mixed with MDSC, so it is difficult to say that only MDSC is selectively lysed. However, in peripheral blood mononuclear cells (PBMC) obtained after removing neutrophils, selective lysis of MDSC can be performed by using anti-CD66c antibodies.

[0036] MDSC-related diseases are diseases that exhibit immunosuppressive activity by MDSCs, and are diseases in which the level of CD66c-positive MDSCs is increased compared to normal cells, which is a criterion for determining the disease. For example, based on the number or activity of CD66c-positive MDSCs in the corresponding normal subject sample per unit volume being 100%, the number or activity of CD66c-positive MDSCs in a subject with a specific disease is about 200% or more, about 300% or more, about 500% or more, about 700% or more, about 1,000% or more, or about 1,500% or more, such as about 200% to 5,000%, or 200% to 3,000%, 200 to 1,500%, etc. For example, an increase in the number of MDSCs can be determined by taking a sample such as blood from a subject suspected of having an MDSC-related disease and a normal subject, analyzing the number of MSDCs in the sample by flow cytometry, and then comparing the number of MDSCs in the subject suspected of having an MDSC-related disease with the number of MDSCs in a normal subject. Specifically, based on the number or activity of MDSCs in a sample per unit volume of a corresponding normal subject being 100%, in a subject suffering from an MDSC-related disease, the number of MDSCs in a sample per unit volume (e.g., blood) is about 200% or more, about 300% or more, about 500% or more, about 700% or more, about 1,000% or more, or about 1,500% or more, for example, about 200% to 5,000%, or 200% to 3,000%, 200 to 1,500%, etc.

[0037] Specifically, MDSC-related diseases are, for example, diseases showing an increase or accumulation of MDSC expressing CD66c in MDSCs with a phenotype of non-lymphoid HLA-DR low / (-), CD11b+ and CD33+, or diseases in which the number of MDSCs is increased compared to the number of normal cells. Examples of MDSC-related diseases include chronic / acute infections, cancers, etc., specifically chronic / acute infections, cancers, etc. showing the immunosuppressive activity of MDSCs. For example, the disease can be chronic / acute infections, cancers, etc. showing an accumulation of CD66c-positive MDSCs in MDSCs with a phenotype of non-lymphoid HLA-DR low / (-), CD11b+ and CD33+.

[0038] MDSC-associated infectious diseases may be infections such as Trypanosoma cruzi, Listeria monocytogenes, Leishmania major, helminths, Candida albicans, Porphyromonas gingivalis or toxoplasmosis or polymicrobial sepsis.

[0039] For example, MDSC-related cancers can be cancers in which CD66c-positive MDSCs increase, including solid cancers and hematological cancers. Examples of solid cancers include colon cancer, fibrosarcoma, thymoma, lung cancer, mesothelioma, lymphoma, prostate cancer, head and neck cancer, melanoma, gastric cancer, liver cancer or breast cancer, or preferably colon cancer, gastric cancer or liver cancer. Preventing, inhibiting or treating cancer and cancer metastasis can, for example, inhibit the growth of cancer cells. Examples of hematopoietic malignancies include acute myeloid leukemia, acute lymphocytic leukemia, acute monocytic leukemia, Hodgkin's lymphoma and non-Hodgkin's lymphoma.

[0040] The present invention relates to an antibody or an antigen-binding fragment thereof that binds to CD66c expressed in MDSC. CD66c (cluster of differentiation 66c), also known as CEACAM6 (carcinoembryonic antigen-related cell adhesion molecule 6) or NCA (non-specific cross-reactive glycoprotein antigen)-90, is known to be an important protein related to cell adhesion. CD66c can be preferably represented by the amino acid sequence of SEQ ID NO: 1 (Genbank Protein No. AAH05008), but is not limited thereto.

[0041] As used herein, the term "antibody" means a substance produced by the stimulation of an antigen in the immune system, and its type is not particularly limited. The antibody can be produced in a non-natural manner, such as recombinant or synthetically. The antibody can be an animal antibody (such as a mouse antibody, etc.), a chimeric antibody, a humanized antibody, or a human antibody. The antibody can be a monoclonal antibody or a polyclonal antibody.

[0042] Anti-CD66c antibodies or antigen-binding fragments thereof specifically bind to a specific epitope of CD66c as described above, and may be selected from the group consisting of animal antibodies (e.g., mouse antibodies), chimeric antibodies, humanized antibodies, and antigen-binding fragments thereof. Animal antibodies may be derived from animal species other than humans, such as rats, mice, goats, guinea pigs, donkeys, rabbits, horses, llamas, camels, birds (e.g., chickens, ducks, etc.), but are not limited thereto. Techniques for producing chimeric antibodies and / or humanized antibodies from such animal antibodies are well known in the art. Humanized antibodies may be of any suitable isotype, such as IgG (IgG1, IgG2, IgG3, IgG4), IgM, IgA, IgD, IgE, or any subclass, preferably IgG1 or IgG2 isotypes, or more preferably defucosylated IgG1 or IgG2 isotypes.

[0043] In addition, unless otherwise indicated, the antibody herein may be understood to include an antigen-binding fragment with antigen-binding ability of an antibody. In this specification, the term "complementarity determining region (CDR)" refers to an antibody region that gives an antibody binding specificity to an antigen in the variable region of an antibody. The antigen-binding fragment of an antibody as described above may be an antibody fragment comprising at least one complementary determining region. The term "CDR (complementarity determining region)" refers to the amino acid sequence of the hypervariable region of the heavy and light chains of an immunoglobulin. Each of the heavy and light chains may include three CDRs (CDRH1, CDRH2, CDRH3 and CDRL1, CDRL2, CDRL3). CDR may provide key contact residues for an antibody to bind an antigen or epitope. On the other hand, in the present disclosure, the meaning of the term "specific binding" or "specific recognition" is the same as that generally known to those skilled in the art.

[0044] The term "antigen-binding fragment" refers to a fragment of an immunoglobulin that is directed against the entire structure thereof, and refers to a polypeptide portion that includes a portion to which an antigen can bind. For example, the fragment may be scFv, (scFv)2, scFv-Fc, Fab, Fab' or F(ab')2, but is not limited thereto.

[0045] The anti-CD66c antibody according to the present invention specifically recognizes and / or binds to CD66c, and the antibody includes a mouse antibody, a chimeric antibody or a humanized antibody. The chimeric antibody in the present invention is an antibody in which the sequence of the variable region is derived from one species, and the sequence of the constant region is derived from another species, for example, the variable region is derived from a mouse, and the constant region is derived from a human. The humanized antibody in the present invention is an antibody with low immunogenicity to humans and has non-human antibody activity. For example, it can be prepared by retaining the non-human CDR region and replacing the rest of the region with a human counterpart. For example, references: Morrison et al., Proc. Natl. Acad. ScL USA, 81: 6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44: 65-92 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988); Padlan, Molec. Immun., 28: 489-498 (1991); Padlan, Molec. Immun., 31(3): 169-217 (1994).

[0046] There is no limitation on the antibody fragments of the present invention, as long as the antibody fragments specifically recognize the CD66c epitope and include a light chain variable region (V L ) and the heavy chain variable region (V H ). The antibody fragment can be selected from the group consisting of Fab, Fab', F(ab')2, scFv, dsFv and CDR. In particular, scFv is obtained by separating the heavy chain variable region (V H ) and light chain variable region (V L ) are connected to a linker polypeptide to prepare a single-chain antibody fragment.

[0047] The term "hinge region" is a region included in the heavy chain of an antibody, present between the CH1 and CH2 regions, and refers to a region that provides flexibility of the antigen binding site in an antibody. For example, the hinge may be derived from a human antibody, and specifically, may be derived from IgA, IgE or IgG, such as IgG1, IgG2, IgG3 or IgG4.

[0048] The anti-CD66c antibody can be a monoclonal antibody or a polyclonal antibody, such as a monoclonal antibody. The monoclonal antibody can be prepared according to methods well known in the art. For example, it can be manufactured using phage display technology.

[0049] Unlike mouse antibodies or chimeric antibodies, humanized antibodies show 10 times higher stability than chimeric 8F5 antibodies in terms of stability, and also have different characteristics that significantly reduce the immunogenicity when administered to humans. Specifically, at high temperatures (e.g., 62°C), the antibody has high stability because the fluorescence variability for ANS reagent is less than 200%.

[0050] Under particularly stringent conditions, due to changes in antibody properties, the chimeric 8F5 antibody showed a 1,406% change in ANS reactivity, while the humanized antibodies showed relatively insignificant changes of about 114% and 133%, indicating that they are remarkably stable proteins.

[0051] Chimeric 8F5 and humanized antibodies enhance T cell activation, which is also shown in T cell activation conditions caused by T cell activators and due to the mixing of allogeneic dendritic cells and T cells from different people. This induction of T cell activation induced cancer cell death when in co-culture conditions with cancer cells, as well as T cell activation under conditions of co-culture with various cancer cells.

[0052] The antibodies or fragments thereof according to the present invention have tumor regression activity and direct inhibitory effects on tumor cell lines. In the present disclosure, tumor regression includes inducing or promoting a reduction in tumor size, and / or inhibiting, stopping or reducing the growth of tumor cells. For example, a reduction in tumor size means that, based on a tumor size of 100% before treatment with a composition comprising an antibody or fragment thereof of the present invention, a tumor size obtained by administering a composition comprising an antibody or fragment thereof is 97% or less, 95% or less, 90% or less, 85% or less, 80% or less, and 75% or less.

[0053] The antibodies according to the present invention have antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), and preferably have ADCC characteristics.

[0054] The antibodies or antigen-binding fragments according to the present invention can improve or treat MDSC-related diseases by combining with natural killer cells or NK cell-derived cell therapy.

[0055] Specifically, the anti-CD66c antibody according to the present invention increases cancer cell killing ability by combining with natural killer cells, and thus the effect of combined treatment with NK cells or NK cell therapeutic agents is excellent for effectively removing not only CEACAM6-positive cancer cells but also CEACAM6-positive MDSCs.

[0056] In specific experiments, as a result of measuring cell viability using the EZ-cytox Enhanced Cell Viability Kit (Daeil Lab), it was confirmed that the apoptotic effect of the combined treatment with natural killer cells was higher in both types of cancer cell lines compared to the case of single treatment ( Fig.12a and 12b ). According to the selective lysis of MDSC caused by the anti-CD66c antibody of the present invention, and the combined effect of the anti-CD66c antibody and NK cells or NK cell therapeutic agents, the antibody can remove targets including CEACAM6-positive cancer cells and CEACAM6-positive MDSC. The anti-CD66c antibody according to the present invention shows ADCC for different target cells (e.g., MDSC and cancer cells), respectively. In the case of cancer patients in which the two types of cells actually increase together, the anti-CD66c antibody of the present invention can remove the two types of targets together, and in combination with the NK cell therapeutic agent, it shows an enhanced efficacy of simultaneously removing cancer cells and MDSC targets.

[0057] The antibody according to the present invention may partially or completely remove fucose as a sugar residue bound to the antibody. The fucose-removed antibody of the present invention has the apoptotic activity of MDSC, and in one embodiment, compared with the antibody in the fucose form, the low-fucose form or the de-fucose form of the antibody of the present invention has the apoptotic activity of MDSC, and therefore has high immune enhancement. As used herein, "normal fucose" or "normal fucose content" refers to an antibody with a fucose content of usually at least 90%. The antibody in the low-fucose form or the de-fucose form according to the present invention can be an antibody with a fucose content of about 10% or less, about 7% or less, or about 5% or less, such as 0 to about 10%, 0 to about 7%, or 0 to about 5%.

[0058] Specifically, the antibodies of the present invention may comprise the following complementarity determining regions (CDRs):

[0059] CDR-H1 comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 9,

[0060] CDR-H2 comprises the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 10,

[0061] CDR-H3 comprising the amino acid sequence of SEQ ID NO: 3,

[0062] CDR-L1 comprises the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 11 or SEQ ID NO: 12,

[0063] CDR-L2 comprising the amino acid sequence of SEQ ID NO: 5, and

[0064] CDR-L3 comprising the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:13.

[0065] The heavy chain variable region of the antibody comprises at least one selected from the group consisting of: a framework sequence (V-FR1) comprising the amino acid sequence of SEQ ID NO: 22, 23, 24, 25, 26 or 27, a framework sequence (V-FR2) comprising the amino acid sequence of SEQ ID NO: 32, 33, 34, 35, 36 or 37, a framework sequence (V-FR3) comprising the amino acid sequence of SEQ ID NO: 42, 43, 44, 45, 46 or 47, and a framework sequence (V-FR4) comprising the amino acid sequence of SEQ ID NO: 52, 53, 54, 55, 56 or 57.

[0066] The light chain variable region of the antibody comprises at least one selected from the group consisting of: a framework sequence (L-FR1) comprising the amino acid sequence of SEQ ID NO: 28, 29, 30 or 31, a framework sequence (L-FR2) comprising the amino acid sequence of SEQ ID NO: 38, 39, 40 or 41, a framework sequence (L-FR3) comprising the amino acid sequence of SEQ ID NO: 48, 49, 50 or 51, and a framework sequence (L-FR4) comprising the amino acid sequence of SEQ ID NO: 58, 59, 60 or 61.

[0067] The antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, 14, 15, 16, 17 or 18, and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8, 19, 20 or 21.

[0068] An example of the mouse antibody or chimeric antibody according to the present invention may be an antibody or an antigen-binding fragment thereof comprising at least one selected from the group consisting of an amino acid sequence of a VH CDR comprising an amino acid sequence of SEQ ID NOs: 1 to 3 and an amino acid sequence of a VL CDR comprising an amino acid sequence of SEQ ID NOs: 4 to 6. CDRs and variable regions of examples of mouse antibodies or chimeric antibodies are summarized in Table 1 below.

[0069] Specifically, examples of the antibody of the present invention may include SEQ ID NO: 1 (CDR1), SEQ ID NO: 2 (CDR2), and SEQ ID NO: 3 (CDR3) as VH CDRs and / or SEQ ID NO: 4 (CDR1), SEQ ID NO: 5 (CDR2), and SEQ ID NO: 6 (CDR3) as VL CDRs.

[0070] The mouse antibody or chimeric antibody may include a VH region comprising the amino acid sequence of SEQ ID NO:7 and a VL region comprising the amino acid sequence of SEQ ID NO:8.

[0071] The present invention relates to a pharmaceutical composition, a kit or a method for preventing or treating MDSC-related diseases and symptoms thereof, comprising mouse antibodies or chimeric antibodies as active ingredients.

[0072] The present invention also relates to a pharmaceutical composition for preventing or treating MDSC-related diseases and their symptoms comprising a mouse antibody or a chimeric antibody as an active ingredient, such as an anti-CD66c antibody or an antigen-binding fragment thereof comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 of an antibody produced by a hybridoma cell, wherein the hybridoma cell is deposited under the deposit number KCLRF-BP-00230. The hybridoma cell was deposited in the Korean Cell Line Research Foundation (KCLRF) on February 22, 2010 as "8F5" and the deposit number is KCLRF-BP-00230, which has been described in detail in KR 10-1214177.

[0073] The present invention can prepare humanized antibodies by using the amino acid sequence of anti-CD66c antibody 8F5 and human framework sequence in mouse antibody or chimeric antibody. Based on the expression degree, aggregation and cell binding degree, the normally expressed humanized antibody is very little aggregated due to the instability of the protein itself, and has the ability of binding target antigen positive cells similar to chimeric antibodies. Specifically, the cell binding spectrum is similar to the cell binding spectrum of chimeric antibodies, and is obtained by multiplying the positive rate (gating %) of antibody positive by the mean fluorescence (mean value), and then the cell binding spectrum is compared with the chimeric antibody to select candidate antibodies within the range of ± 20% (Example 2). Therefore, when the CDR region sequence of mouse antibody is inserted into the framework region of human antibody when preparing humanized antibodies, due to the change of the original protein structure, the binding ability of the prepared antibody is rapidly reduced. Considering that the binding ability of the prepared antibody is reduced, the selected humanized antibody of the present invention is a very excellent antibody.

[0074] Preferably, five types of recombinant humanized antibodies showing high binding affinity based on cell binding ability compared with the chimeric antibody are selected, and the five types of recombinant humanized antibodies are assayed for binding to CD66c antigen and antigens similar to CD66 antigen by ELISA.

[0075] In addition, compared to chimeric antibodies, the humanized antibodies according to the present invention exhibit excellent stability, such as antibodies with stability reflected as a change in ANS reactivity of less than 200%. A change in ANS reactivity of less than 200% is considered to be a very small change, while a change value of more than 200% can be interpreted as the observation of ANS reactivity due to significant structural changes in the protein. Therefore, the humanized antibodies according to the present invention have antigen binding activity and cell binding ability similar to those of chimeric antibodies, and the physical stability of the antibody protein itself is increased, which can be very excellent in terms of the pharmaceutical applicability of therapeutic antibodies.

[0076] The fluorescence change of the antibody against the ANS agent can be measured by dividing the difference between the fluorescence value measured under low temperature conditions (eg, 4° C.) and the fluorescence value measured under high temperature conditions (eg, 62° C.) by the fluorescence value measured under low temperature conditions.

[0077] [Mathematical equation]

[0078] Fluorescence change = (fluorescence value measured under high temperature conditions - fluorescence value measured under low temperature conditions) / (fluorescence value measured under low temperature conditions)

[0079] As a method for obtaining a specific fluorescence change of an antibody, after standing for 4 hours under refrigerated conditions (4°C) and at a temperature of 62°C, the reactivity of the ANS reagent is measured by a fluorescence reader and expressed as a fluorescence value, and the fluorescence change can be obtained using this equation.

[0080] Examples of humanized antibodies according to the present invention may include one or more amino acid sequences selected from the group consisting of: amino acid sequences of CDRs of heavy chain variable regions or light chain variable regions that determine the amino acid sequences of SEQ ID NOs: 9 to 13. Examples of mouse antibodies and chimeric antibodies may include one or more amino acid sequences selected from the group consisting of: amino acid sequences of CDRs of heavy chain variable regions or light chain variable regions that determine the amino acid sequences of SEQ ID NOs: 1 to 3 or SEQ ID NOs: 4 to 6.

[0081] Specifically, examples of humanized antibodies include determining the amino acid sequence of CDR1 of the VH region comprising the amino acid sequence of SEQ ID NO: 1 or 9, determining the amino acid sequence of CDR2 of the VH region comprising the amino acid sequence of SEQ ID NO: 2 or 10, and determining the amino acid sequence of CDR3 of the VH region comprising the amino acid sequence of SEQ ID NO: 3.

[0082] Examples of humanized antibodies include those whose amino acid sequence is determined to be CDR1 of a VL region comprising the amino acid sequence of SEQ ID NO: 4, 11 or 12, whose amino acid sequence is determined to be CDR2 of a VL region comprising the amino acid sequence of SEQ ID NO: 5, and whose amino acid sequence is determined to be CDR3 of a VL region comprising the amino acid sequence of SEQ ID NO: 6 or 13.

[0083] Examples of the humanized antibody include a heavy chain variable region selected from the group consisting of the amino acid sequences of SEQ ID NO: 7 and SEQ ID NOs: 14 to 18 and a light chain variable region selected from the group consisting of the amino acid sequences of SEQ ID NO: 8 and SEQ ID NOs: 19 to 21, but excluding antibodies containing SEQ ID NO: 7 and SEQ ID NO: 8.

[0084] CDR sequences and variable region sequences according to examples of humanized antibodies are summarized in Table 1 below.

[0085] [Table 1]

[0086]

[0087]

[0088] The framework sequences of one example of the humanized antibody according to the present invention are shown in Tables 2 and 3 below, wherein the antibody may comprise at least one selected from the group consisting of Frameworks 1 to 4 of the heavy chain variable region and Frameworks 1 to 4 of the light chain variable region, and may be an antibody comprising one or more frameworks.

[0089] Specifically, in the heavy chain variable region, the amino acid sequence of framework 1 may include SEQ ID NOs: 23 to 27, the amino acid sequence of framework 2 may include SEQ ID NOs: 32 to 37, the amino acid sequence of framework 3 may include SEQ ID NOs: 43 to 47, and the amino acid sequence of framework 4 may include SEQ ID NOs: 53 to 57.

[0090] In the light chain variable region, the amino acid sequence of framework 1 may include SEQ ID NOs: 29 to 31, the amino acid sequence of framework 2 may include SEQ ID NOs: 39 to 41, the amino acid sequence of framework 3 may include SEQ ID NOs: 49 to 51, and the amino acid sequence of framework 4 may include SEQ ID NOs: 59 to 61. The following table shows framework sequences according to examples of humanized antibodies.

[0091] [Table 2]

[0092]

[0093] [Table 3]

[0094]

[0095]

[0096] The humanized antibody may comprise a VH region selected from the group consisting of the amino acid sequences of SEQ ID NOs: 14 to 18 and a VL region selected from the group consisting of the amino acid sequences of SEQ ID NOs: 19 to 21. Specifically, examples of humanized antibodies include: an antibody (Vk8+VH6) comprising a VH region comprising an amino acid sequence of SEQ ID NO: 15 and a VL region comprising an amino acid sequence of SEQ ID NO: 21; an antibody (Vk8+VH11) comprising a VH region comprising an amino acid sequence of SEQ ID NO: 18 and a VL region comprising an amino acid sequence of SEQ ID NO: 21; an antibody (Vk5+VH7) comprising a VH region comprising an amino acid sequence of SEQ ID NO: 16 and a VL region comprising an amino acid sequence of SEQ ID NO: 19; an antibody (Vk7+VH6) comprising a VH region comprising an amino acid sequence of SEQ ID NO: 17 and a VL region comprising an amino acid sequence of SEQ ID NO: 20; an antibody (Vk7+VH10) comprising a VH region comprising an amino acid sequence of SEQ ID NO: 15 and a VL region comprising an amino acid sequence of SEQ ID NO: 20; an antibody (Vk7+VH7) comprising a VH region comprising an amino acid sequence of SEQ ID NO: 15 and a VL region comprising an amino acid sequence of SEQ ID NO: 20 NO: 16 and a VL region containing the amino acid sequence of SEQ ID NO: 20; an antibody (Vk7+VH5) comprising a VH region containing the amino acid sequence of SEQ ID NO: 14 and a VL region containing the amino acid sequence of SEQ ID NO: 20; and an antibody (Vk8+VH7) comprising a VH region containing the amino acid sequence of SEQ ID NO: 16 and a VL region containing the amino acid sequence of SEQ ID NO: 21. Specific combinations and amino acid sequences of the antibodies are shown in Table 6 below. Preferred examples of the antibody include: an antibody (Vk8+VH6) comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and a VL region comprising the amino acid sequence of SEQ ID NO: 21; an antibody (Vk8+VH11) comprising a VH region comprising the amino acid sequence of SEQ ID NO: 18 and a VL region comprising the amino acid sequence of SEQ ID NO: 21; an antibody (Vk5+VH7) comprising a VH region comprising the amino acid sequence of SEQ ID NO: 16 and a VL region comprising the amino acid sequence of SEQ ID NO: 19; an antibody (Vk7+VH6) comprising a VH region comprising the amino acid sequence of SEQ ID NO: 17 and a VL region comprising the amino acid sequence of SEQ ID NO: 20; and an antibody (Vk7+VH10) comprising a VH region comprising the amino acid sequence of SEQ ID NO: 15 and a VL region comprising the amino acid sequence of SEQ ID NO: 20.

[0097] Anti-CD66c antibodies or fragments thereof can be coupled with various labeling agents, toxins or anti-tumor drugs. It is obvious to those skilled in the art that the antibodies of the present invention can be coupled with labeling agents, toxins or anti-tumor drugs by methods well known in the art. Such coupling can be performed chemically at the attachment site after the antibody or antigen is expressed. Alternatively, the coupling product can be engineered into the antibody or antigen of the present invention at the DNA level. Subsequently, the product can be expressed in a suitable host system as described below, and the expressed protein is collected, and if necessary, the protein is renatured. Coupling can be performed by joints known in the art. In particular, various joints that release toxins or anti-tumor drugs under acidic or reducing conditions or after exposure to specific proteases can be used for this technology. In some embodiments, it may be desirable for the joint to be attached to the labeling agent, toxin or anti-tumor drug through a spacer arm of various lengths to reduce potential steric hindrance.

[0098] Using CD66c protein, CD66c antigenic determining region, CD66c part containing CD66c antigenic determining region or cells expressing CD66c antigenic determining region as antigen, using common methods, antibodies or fragments thereof against the antigenic determining region of CD66c can be produced. For example, the method for producing anti-CD66c antibodies can be achieved by preparing a cell line that produces anti-CD66c antibodies, the method comprising: (a) injecting and immunizing animals with CD66c protein, CD66c antigenic determining region, CD66c part containing CD66c antigenic determining region or cells expressing CD66c antigenic determining region, (b) obtaining spleen cells that produce antibodies specific to CD66c, and (c) fusing spleen cells with myeloma cells to obtain hybridoma cells, and selecting hybridoma cells that produce antibodies against CD66c. Antibodies can be isolated by culturing cell lines in vitro or introducing cell lines in vivo. For example, the cell line can be injected intraperitoneally into mice, and then the antibodies are isolated and purified from the ascites. Isolation and purification of the monoclonal antibody can be achieved by subjecting the culture supernatant and ascites fluid to ion exchange chromatography (DEAE or DE52) or affinity chromatography using an anti-immunoglobulin column or a protein A column.

[0099] The antigenic determinant region bound by the antibody of the present invention exhibits MDSC-specific expression. Therefore, the anti-CD66c antibody can not only be effectively used to detect MDSC, but also can exert cytotoxicity only on tumor cells when the antibody carries a toxic substance.

[0100] Another embodiment provides the use of the anti-CD66c antibody according to the present invention as a marker for detecting MDSC, or specifically, the use of the anti-CD66c antibody or its antigen-binding fragment to detect MDSC, diagnose MDSC-related diseases or provide information on the diagnosis of MDSC-related diseases.

[0101] For example, it provides a composition for detecting MDSC by using an anti-CD66c antibody or an antigen-binding fragment thereof, the composition comprising a substance that interacts with the antigenic determining region of the antibody. The interacting substance includes all substances that can interact with the antigenic determining region CD66c, and can be at least one selected from small molecule chemicals, antibodies, antigen-binding fragments of antibodies, aptamers, etc.

[0102] By contacting the sample with the antibody of the present invention and determining the presence of CD66c in the sample, the diagnostic composition of the present invention is used to detect the undesirable expression or overexpression of CD66c in various cells, tissues or another suitable sample. Therefore, the diagnostic composition of the present invention can be used to assess the onset or state of the disease, as defined below. In particular, MDSCs capable of expressing CD66c can be targeted with the antibodies of the present invention or fragments or derivatives thereof. Cells to which the antibodies of the present invention are bound may be attacked by immune system functions (e.g., complement systems) or cell-mediated cytotoxicity, thereby reducing the number of cells showing undesirable expression or overexpression of CD66c or completely eradicating the cells.

[0103] As a specific example, a method or composition for diagnosing MDSC-related diseases using the antibody or antigen-binding fragment against CD66c according to the present invention is provided.

[0104] In the case of diagnosing MDSC-related diseases (such as cancer), the anti-CD66c antibody or its antigen-binding fragment according to the present invention can be used for diagnosis and treatment by targeting MDSC infiltrating around cancer tissues (regardless of the expression of CEACAM6 antigen in cancer tissues or cancer cells). The anti-CD66c antibody according to the present invention not only binds to CD66c expressed in solid cancer cells, but also binds to CD66c expressed in MDSC. Therefore, even in cancers that do not express CD66c in solid cancer cells, the antibody can detect cancer by targeting the increased state of MDSC caused by cancer. Specifically, in cancer tissues of lung adenocarcinoma that are CEACAM6-positive in cancer cells, and lung squamous cell carcinoma, urinary bladder cancer, and malignant melanoma that are CEACAM6-negative in cancer cells, the results of staining the cancer tissues confirmed that CEACAM6-positive MDSCs are present in non-tumor sites of cancer tissues ( Fig.13 ).

[0105] Thus, cancer patients show elevated MDSC levels regardless of whether CEACAM6 is positive on the surface of cancer cells, and thus, as shown in the results of Example 8, MDSC infiltrating into the cancer microenvironment can be detected and confirmed. When considered together with the results of Example 5.2 showing that MDSC can be selectively solubilized, this suggests that MDSC can be used as a target for diagnostic and therapeutic purposes regardless of whether CEACAM6 is positive on cancer cells. Although the presence or absence of CEACAM6 expression on the surface of cancer cells can vary depending on the type of cancer, MDSC is increased in most cancer types regardless of whether CEACAM6 is expressed or not. Therefore, the anti-CD66c antibodies according to the present invention can target MDSC and can be used for diagnostic and therapeutic purposes in a variety of applications.

[0106] In another embodiment, the antibody of the present invention or its fragment or derivative is coupled to a labeling agent. Such an antibody is particularly suitable for diagnostic applications.

[0107] The composition of the present invention may be administered alone as an active agent or in combination with other agents.

[0108] Yet further embodiments of the present invention relate to methods for detecting MDSC, comprising (a) reacting an anti-CD66c antibody with a sample comprising MDSC, and (b) determining that the sample is MDSC if the sample is positive for the antibody. The sample may include, but is not limited to, lymph, bone marrow, blood, and blood cells. When used to screen for MDSC, the anti-CD66c antibody may be conjugated to a marker that can indicate antigen-antibody reactivity. Markers for this purpose may include radioisotopes, fluorescent substances, luminescent substances, chromogens, and dyes.

[0109] Likewise, the anti-CD66c antibodies of the present invention can be provided as a kit for diagnosing MDSC-related diseases. In addition to the anti-CD66c antibodies, the diagnostic kit may also include means for detecting antigen-antibody reactions. The detection means may be an agent for performing a technique selected from the group consisting of: flow cytometry, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescent immunoassay (FIA) and luminescent immunoassay (LIA).

[0110] The therapeutic effect of solid cancer is the effect of inhibiting the progression of cancer, including not only the growth inhibition (reduction in amount) and apoptosis effect of cancer cells (especially cancer stem cells) or cancer tissues including cancer cells, but also the inhibition of migration, invasion, metastasis, etc. In order to maximize the effect of the antibody according to the present invention, the antibody can be treated in combination with a STING agonist or 5-Fu, and a higher effect can be expected in the combined treatment.

[0111] As used herein, the term "subject" or "patient" refers to a mammal, including primates (e.g., humans, monkeys, etc.) and rodents (e.g., mice, rats, etc.), who suffer from or have the possibility of suffering from an MDSC-related disease or symptom and therefore need to alleviate, prevent and / or treat MDSC.

[0112] The administration of the antibody or fragment thereof according to the present invention can be carried out in any acceptable manner. For example, a therapeutic agent comprising an anti-CD66c antibody as an active ingredient is administered orally or parenterally, preferably parenterally to a subject, such as a human or animal, with a MDSC-related disease. The therapeutic agent may include a pharmaceutically acceptable excipient, and the dosage of the therapeutic agent may vary depending on the patient's condition, and may be in the range of, for example, 3 mg to 6,000 mg per day. The therapeutic agent may take the form of, for example, a liquid, a powder, an emulsion, a suspension, or an injection, but is not limited thereto.

[0113] Further, the present invention provides a method for treating MDSC-related diseases using at least one selected from an antibody against the antigenic determining region of CD66c, a fragment of the antibody (F(ab')2, Fab, Fv, etc.) and a ligand against the antigenic determining region of CD66c. The antibody or its fragment may be monoclonal or polyclonal and may be derived from humans or animals. The anti-CD66c antibody or its fragment may further comprise the above-mentioned toxin. The toxin may be fused, coupled, conjugated or linked to the antibody using well-known techniques.

[0114] The pharmaceutical composition of the present invention can be used as a single active agent or in combination with any other agent preferably used to treat the target disease. In addition, the antibodies of the present invention can be used in combination with other anticancer therapies (e.g., chemotherapy, radiotherapy, cell therapy, etc.). Various well-known anticancer agents can be used in chemotherapy or cell therapy.

[0115] Technical Effects

[0116] The present invention provides an immunopotentiator and use of the immunopotentiator for preventing, improving or treating MDSC-related diseases. The immunopotentiator comprises an antibody or an antigen-binding fragment thereof that specifically binds to CD66c expressed in myeloid-derived suppressor cells (MDSCs). BRIEF DESCRIPTION OF THE DRAWINGS

[0117] Figure 1 The results are shown in which an antibody gene was cloned from mouse 8F5 antibody and expressed as a chimeric recombinant antibody, which then bound to the surface of CD66c antigen-positive A549 cells.

[0118] Figures 2a to 2cThe results of HPLC analysis of 8 recombinant humanized antibodies selected first in 96 recombinant humanized antibodies are shown. For each antibody, the result shown on the left is measured at OD 220nm, and the result shown on the right is measured at OD 280nm. The result indicates whether the antibody and the impurities (e.g., fragments) derived from the antibody are aggregated.

[0119] Figures 3a to 3e A similar degree of cell surface binding of the recombinant humanized antibodies was shown compared to the chimeric antibodies, and thus surface binding of CD66c antigen-positive cells of 8 recombinant humanized antibodies selected first from among 96 recombinant humanized antibodies was confirmed.

[0120] Figure 4a and 4b The results of ELISA analysis of the binding ability of 5 recombinant humanized antibodies selected from 96 recombinant humanized antibodies to the CD66c antigen are shown. Figure 4a Results showing CECACAM6 (CD66c) as antigen, Figure 4b Results for the CEACAM1 (CD66a) antigen are shown.

[0121] Figure 5a and 5b Shown are the results of evaluating the antibody stability of 5 recombinant humanized antibodies selected from 96 recombinant humanized antibodies under harsh temperature conditions.

[0122] Figures 6a to 6d Cell surface binding of the recombinant humanized antibody expressed in CHO cells to CD66c antigen-positive cells A549 is shown.

[0123] Figure 7 It is a schematic diagram for helping to understand the MDSC analysis method. Specifically, the upper figure is a schematic diagram of a specific dot plot obtained by indicating the monocyte area and granulocyte area other than lymphocytes according to cell size in the dot plot, selecting a group with no or low expression of HLA-DR, and determining a group with CD11b and CD33 positive in the group as MDSC. The lower figure is the result of the positive rate of DNP002 in the determined MDSC group.

[0124] Figure 8 Representative results analyzing the killing effect of MDSCs after treatment with DNP002, showing significant reduction of MDSCs by defucosylated DNP002. CD66b is expressed in granulocytic MDSCs but not in monocytic MDSCs and is used for MDSC subtype classification. Figure 8 Most of the MDSCs noted in the Figure 1 were CD66b positive and could be classified as granulocytic MDSCs. Thus, granulocytic MDSCs were significantly reduced by treatment with DNP002.

[0125] Fig. 9 It is the result of analyzing the MDSC killing effect after treatment with DNP002, showing the comparison results of the percentage of MDSC number reduced due to DNP002 treatment in 5 patients compared with the control group, where P#1 on the horizontal axis in the figure represents the patient's whole blood #1, and the vertical axis represents the change in the relative MDSC activity %.

[0126] Fig.10 The results of analyzing the MDSC-killing effect by flow cytometry after treating PBMCs isolated from the blood of gastric cancer patients with the DNP002 antibody are shown.

[0127] Fig.11a The results of comparing the MDSC-killing effects of DNP002 according to the isotype confirmed that DNP002 of the defucosylated IgG1 type most effectively induced MDSC-killing in the blood.

[0128] Fig.11b The results of comparing the MDSC killing effect according to the isotype of DNP002 in 5 gastric cancer patients show that the defucosylated IgG1 type DNP002 obtained by comparing the percentage of MDSC killing effect provides the highest MDSC killing effect in the blood of 5 gastric cancer patients, where P#1 on the horizontal axis in the figure represents the patient's whole blood #1, and the vertical axis represents the change in the relative MDSC activity %.

[0129] Fig.12a and Figure 12b The results are as follows: the apoptotic effect of DNP002 antibody alone, NK cells alone, and a combination of DNP002 antibody and NK cells was analyzed on the CEACAM6-positive gastric cancer cell line A549 and the pancreatic cancer cell line AsPC-1 as target antigens.

[0130] Fig.13 The diagram shows the presence of CEACAM6-positive MDSCs in non-tumor sites of cancer tissues by immunostaining lung adenocarcinoma in which cancer cells are positive for CEACAM6, and lung squamous cell carcinoma, urinary bladder cancer, and skin cancer (malignant melanoma) in which cancer cells themselves are negative for CEACAM6, using CEACAM immunostaining. DETAILED DESCRIPTION

[0131] The present invention may be better understood through the following examples, which are set forth for purposes of illustration but are not to be construed as limiting the present invention.

[0132] Example 1: Preparation of anti-CD66c chimeric antibody

[0133] 1.1. Cloning of anti-CD66c antibody gene sequence

[0134] The 8F5 antibody gene was cloned using a mouse Ig primer set (Millipore, catalog number: 69831). RNA isolated from the 8F5 hybridoma was subjected to PCR using a mouse Ig primer set, inserted into a pGem-T vector (Promega, catalog number: A3600), sequenced to confirm the DNA sequence, and the mouse antibody gene was identified by the IMGT site (www.imgt.org). The heavy chain variable region sequence and light chain variable region sequence of the 8F5 antibody analyzed are as follows.

[0135] [Table 4]

[0136]

[0137]

[0138] 1-2. Production of chimeric antibodies

[0139] Based on the amino acid sequence of the constructed anti-CD66c mouse antibody 8F5, an anti-CD66c chimeric antibody was prepared.

[0140] 1-2-1. Plasmid production

[0141] For the expression of anti-CD66c chimeric antibody, heavy chain expression plasmid and light chain expression plasmid were prepared separately. POptiVEC (Invitrogen) vector was used as the light chain expression plasmid, and pcDNA3.3 (Invitrogen) vector was used as the heavy chain expression plasmid.

[0142] In order to express the variable region encoding cDNA and constant region encoding cDNA of each antibody as a continuous amino acid sequence without inserting additional amino acids, the coding sequence of the cloned variable region and the known coding sequence of the human IgG1 constant region (heavy chain) and kappa constant region (light chain) were synthesized (Bioneer). The synthesized heavy chain gene and light chain gene were cut with restriction enzymes Xho I and Sal I, and the light chain gene fragment was connected to the pOptiVec vector and the heavy chain gene fragment was connected to the pcDNA3.3 vector to construct a complete antibody expression plasmid (pcDNA3.3-anti-CD66c heavy chain expression plasmid and pOptiVEC-anti-CD66c light chain expression plasmid).

[0143] 1-2-2. Transfection

[0144] The prepared pcDNA3.3-anti-CD66c heavy chain expression plasmid and pOptiVEC-anti-CD66c light chain expression plasmid were transfected into CHO cell-derived DG44 cells (Invitrogen).

[0145] Three days before transfection, DG44 cells in suspension were adapted to MEMS medium containing 5% FBS to convert the cells into adherent cells and improve transfection efficiency. Transfection was performed on 6-well plates using ViaFect transfection reagent (Promega, catalog number: E4981). One day before transfection, 1×10 5 DG44 cells adapted to the adherent state were prepared by passage at a concentration of 10 cells / well. The amount of DNA used for transfection was determined by combining 2ug and 1.5ug of pcDNA3.3-anti-CD66c heavy chain expression plasmid and pOptiVEC-anti-CD66c light chain expression plasmid at a ratio of 1.5:1. Transfection was performed for 48 hours. The transfected cell population was analyzed by flow cytometry. Figure 1 As shown, the expression of the chimeric antibody was confirmed by the A549 non-small cell lung cancer cell line. Figure 1 The results are shown in which an antibody gene was cloned from mouse 8F5 antibody and expressed as a recombinant chimeric antibody, which then bound to the surface of CD66c antigen-positive A549 cells.

[0146] Example 2: Preparation of humanized anti-CD66c monoclonal antibody

[0147] 2.1 Selection of recombinant antibody sequences by computer simulation humanization

[0148] By maintaining the heavy and light chain CDRs of the mouse anti-CD66c antibody, 8F5 (heavy chain amino acid sequence: SEQ ID NO: 7, heavy chain encoding DNA: SEQ ID NO: 62; light chain amino acid sequence: SEQ ID NO: 8, heavy chain encoding DNA: SEQ ID NO: 63) as similar as possible (CDRH1: ASGYSFTDYTMN) SEQ ID NO: 1, CDRH2: SEQ ID NO: 2 (LINPFHGGTVSNQRFKV); CDRH3: SEQ ID NO: 3 (VRGDPVRHYYALAY); CDRL1: SEQ ID NO: 4 (GASENVYGTL); CDRL2: SEQ ID NO: 5 (GATNLAD); CDRL3: SEQ ID NO: 6 (VATYYCQNVLSAPYT), if the antigen binding ability is equal to or higher, the humanized antibody sequence obtained by recombination of the site sequences of the framework region is selected by a computer simulation method based on the germline sequence encoding the human antibody gene. As shown in Table 5, the human antibody germline genes used as the framework of the recombinant humanized antibody sequence are most similar to the heavy chain and light chain of the mouse CD66c antibody 8F5, respectively. Table 6 shows the amino acid sequence and nucleic acid sequence of the heavy chain variable region and light chain variable region of the mouse CD66c antibody and the CDR sequence of the heavy chain variable region and light chain variable region.

[0149] [Table 5]

[0150]

[0151] Using human antibody germline gene sequences, 12 heavy chain variable regions and 8 light chain variable regions were selected as the selected humanized 8F5 antibody sequences, as shown in Table 3. Tables 6 to 8 show the amino acid sequences, CDR sequences and framework sequences of the heavy chain variable regions and light chain variable regions of the selected humanized antibodies. The heavy chain variable regions and light chain variable regions of chimeric antibodies and humanized antibodies are shown in Table 1. Preferably, mouse antibodies and humanized antibodies have the same amino acid sequences of heavy chain CDR3 and light chain CDR2. The bold and underlined portions in Table 6 are the CDR sequences of antibodies. The bold and underlined portions in Table 7 indicate modified amino acids.

[0152] [Table 6]

[0153]

[0154]

[0155] [Table 7]

[0156]

[0157] [Table 8]

[0158]

[0159]

[0160] 2.2 Expression and analysis of recombinant humanized antibodies

[0161] The selected antibody sequences were expressed in the form of human IgG1 by connecting the human IgG1 heavy chain constant region and the kappa light chain constant region, respectively, in 293 cells. Seven days after transfection, the recombinant humanized antibodies were purified using KanCap A resin (Kaneca).

[0162] Purified antibodies were quantified by measuring at OD 280 nm and subjected to SDS-PAGE. Antibody purity and aggregation were analyzed by HPLC using a Sepax Zenix-C SEC-300 size exclusion chromatography column (Sepax Technologies) and by analyzing at 280 nm and 220 nm. Figures 2a to 2c ).

[0163] 2.3 Cell binding and antigen binding analysis of recombinant humanized antibodies

[0164] 2-3-1 Cell binding assay

[0165] Each of the 96 expressed recombinant humanized antibodies was poured into a test tube containing an equal amount (1 ug) of CD66c-positive A549 non-small cell lung cancer cell line, reacted at 4°C for 30 minutes, washed with PBS, and FITC-conjugated goat anti-human IgG (DiNona, Korea) was added and incubated at 4°C for 15 minutes for treatment. After washing with PBS, the cells were analyzed by flow cytometry (Stratedigm, S1000EXi), and the results are shown below.

[0166] Among the 96 recombinant humanized antibody candidates, 8 were first selected based on the degree of expression, presence of aggregation and degree of cell binding (Tables 9 and 10, Figures 3a to 3e ). Tables 9 and 10 are the analysis results of the initially selected anti-CD66c humanized antibodies and chimeric 8F5, and Table 10 shows the results of flow cytometry analysis.

[0167] [Table 9]

[0168]

[0169] [Table 10]

[0170]

[0171] Table 9 shows the expression degree, presence or absence of aggregation and degree of cell binding of 8 selected antibodies. Specifically, the expression level and molecular weight of 8 selected antibodies are summarized. In addition, according to the results of flow cytometry in Table 10, it was confirmed that 8 recombinant humanized antibodies showed a cell binding strength of ± 20%, which showed a cell binding strength very similar to that of chimeric antibodies. As a result, 8 antibodies were first selected from 96 humanized candidate antibodies, and the 8 antibodies were normally expressed, the aggregation formed due to the instability of the protein itself was small, and the binding affinity to target antigen-positive cells was similar to that of chimeric antibodies.

[0172] In particular, the binding ability of cell lines is different in value. However, as shown in Figure 3, the actual cell binding spectrum is similar to that of chimeric antibodies, and by multiplying the antibody positive rate (gating %) by the average fluorescence (mean value) and obtaining a numerical value compared with chimeric antibodies, humanized antibodies within ± 20% are selected, and 8 kinds of humanized antibodies can be determined. Generally, when producing humanized antibodies, when the mouse antibody CDR region sequence is inserted into the framework region of the humanized antibody, the antibody binding affinity is sharply reduced due to the change of the original protein structure. Taking into account the general properties of humanized antibodies, very good humanized antibodies have been selected in the present invention.

[0173] 2-3-2 Antigen binding assay

[0174] Among the selected 8 recombinant humanized antibodies, 5 recombinant humanized antibodies showing high binding affinity compared with the chimeric antibody were selected, and the binding affinities of the 5 recombinant humanized antibodies to CD66c antigen and similar CD66 antigen were analyzed by ELISA, respectively.

[0175] [Table 11]

[0176] Protein ID HC&LC combination 3043 Vk8+VH6 3058 Vk8+VH11 2938 Vk5+VH7 3007 Vk7+VH6 3019 Vk7+VH10

[0177] Antigens CD66c (CEACAM6; Sino Biological) and CEACAM1 antigens (Sino Biological) were coated on 96-well plates at a ratio of 100 ng per well and then blocked. The primary antibody was diluted 3-fold from 10 ug / ml and allowed to bind for 1 hour at 37°C. The primary antibody was diluted 3-fold from the initial concentration of 10 ug / ml and allowed to bind for 1 hour at 37°C. Goat anti-human Ig-HRP conjugate (Jackson ImmunoResearch) as the secondary antibody was diluted 1:10,000 and incubated at 37°C for 30 minutes. Three washes were performed between each step, and then the TMB reaction was performed with an equal amount of 1N H 2 SO 4The solution (100 ul) was stopped and then the OD value was measured at 450 nm.

[0178] As the experimental results, Figure 4a , Table 12, Figure 4b The binding affinities of 5 recombinant humanized antibodies selected from 96 recombinant humanized antibodies to CD66c antigen are shown in Table 13. Figure 4a and Table 12 shows the binding ability of the antibodies to the CECACAM6 (CD66c) antigen, Figure 4b and Table 13 are the results for CEACAM1 (CD66a) antigen.

[0179] from Figure 4a , Table 12, Figure 4b Judging from the binding affinities of antibodies to antigens in Table 13, all anti-CECACAM6 antibodies showed similar binding profiles to the anti-CEACAM6 chimeric antibodies and were divided into the group that did not bind to CEACAM1 or weakly bound to CEACAM1.

[0180] [Table 12]

[0181]

[0182] [Table 13]

[0183]

[0184] 2.4 Stability analysis of recombinant humanized antibodies

[0185] The stability of the antibodies was determined by placing the five recombinant humanized antibodies selected by their binding profiles to antigens and cells in Example 3.3 under high temperature conditions.

[0186] Stability was determined by binding experiments using 8-anilino-1-naphthalenesulfonic acid (ANS, Sigma), a compound that can detect protein denaturation by measuring the change in fluorescence wavelength between binding and non-binding to the exposed hydrophobic sites when the protein is denatured.

[0187] The concentration of the recombinant humanized antibody was adjusted to 0.2 mg / ml using PBS (phosphate buffered saline) and placed at 50°C for 4 hours under harsh conditions. Each 500 μl of the diluted solution of the antibody to be analyzed was mixed with 20 μl of 0.2 μg / ml ANS solution and analyzed after 5 minutes using a fluorescence plate reader under conditions of 360 nm excitation and 460 nm emission. In addition, the ANS reagent reaction was measured at 70°C for another 30 minutes.

[0188] Figure 5a and 5bThe results of confirming the antibody stability of the five recombinant humanized antibodies shown in Table 11 under harsh temperature conditions are shown. That is, after the antibodies were placed at 50°C for 4 hours under harsh conditions and then at 70°C for 30 minutes, the reactivity of the ANS reagent was measured by fluorescence. Figure 5a The experimental results show that when the antibodies were placed at 50°C for 4 hours, most of the 5 antibodies showed little ANS response, but when the antibodies were placed at 70°C for another 30 minutes, the fluorescence value increased greatly. Among them, the recombinant antibody with protein ID: 3058 showed the smallest increase in fluorescence value, thus showing the most stable property in temperature changes among the 5 recombinant humanized antibodies.

[0189] In order to measure the change in the reactivity of the ANS reagent, the antibody was placed under refrigerated conditions (4±2°C) and 62°C for 4 hours each, and the reactivity of the ANS reagent was analyzed using a fluorescence plate reader in the same manner as described above. The change in the fluorescence value of the antibody against the ANS reagent can be determined by obtaining the difference between the fluorescence value measured under low temperature conditions (e.g., 4°C) and the fluorescence value measured under high temperature conditions (e.g., 62°C) and dividing it by the fluorescence value measured under low temperature conditions.

[0190] [Mathematical equation]

[0191] Fluorescence value change = (fluorescence value measured under high temperature conditions - fluorescence value measured under low temperature conditions) / (fluorescence value measured under low temperature conditions)

[0192] like Figure 5bAs shown, the reaction was carried out for 4 hours at a temperature of 62°C, which was increased relative to the temperature (50°C), and then the reactivity of the ANS reagent was confirmed. The five recombinant humanized antibodies and chimeric antibodies had almost no ANS reaction under refrigerated conditions, but increased with increasing temperature. However, the chimeric 8F5 antibody showed that the reactivity of the ANS reagent increased to 1,406% by maintaining it under a temperature condition of 62°C, and a precipitate was produced, showing an unstable result. However, the five humanized antibodies showed a change in the reactivity of the ANS reagent that was significantly lower than that of the chimeric antibody, and no precipitate was produced. In particular, the changes in the reactivity of the ANS reagent of the humanized antibody protein ID 3019 and protein ID 3058 were 114% and 133%, respectively, and were therefore considered to be the most stable antibodies. The increase in the reactivity of the ANS reagent means that the exposure of the hydrophobic amino acids placed inside the protein structure is increased, which causes the denaturation of the protein structure and leads to the formation of protein aggregates (i.e., precipitates). The humanized antibodies according to the present invention are considered to be stable antibodies with a change in ANS reactivity of less than 200%. A change in ANS reactivity of less than 200% is considered to be very low, and above this value it is considered that the protein structure has a more significant change and ANS reactivity is observed. Therefore, the fact that the humanized antibody according to the present invention has antigen binding ability and cell binding ability similar to those of the chimeric antibody and increased physical stability of the antibody protein itself is a very excellent feature in terms of the pharmaceutical usability of the therapeutic antibody.

[0193] 2.5 CHO cell expression and analysis of recombinant humanized antibodies

[0194] 5 kinds of recombinant humanized antibodies selected in embodiment 2.3 are expressed in CHO cells for expressing most therapeutic antibodies and the antibodies are analyzed. The light chain variable region DNA sequence and the heavy chain variable region DNA sequence of 5 kinds of recombinant humanized antibodies selected by construction are codon optimized, synthesized, and connected to human IgG1 constant region gene by overlapping PCR method. The product is cut with XhoI and EcoRI, and connected to pcDNA3.4 carrier (Life Technology company). Table 11 shows the light chain and heavy chain combination of humanized antibodies selected for CHO cell expression.

[0195] The DNA primer sequences used for PCR on the variable and constant region genes are shown in Table 14 below.

[0196] [Table 14]

[0197]

[0198] Five recombinant humanized antibodies were transiently transfected using ExpiCHO (trademark) expression system reagent (ThermoFisher, catalog number: A29133), and the expressed antibodies were transfected into CD66c-positive A549 non-small lung cancer cell line and analyzed by flow cytometry, as shown in FIG. Figures 6a to 6c All five recombinant humanized antibodies showed similar binding affinities to the chimeric antibody. The fluorescence value measured by the flow cytometer was divided by the antibody expression level in the CHO culture medium to obtain the antibody binding affinity relative to the antibody expression level, as shown in Figure 6d As shown. Therefore, it was confirmed that the recombinant humanized antibody was appropriately expressed in CHO cells. The binding affinity of the antibody to the cell surface was determined to be 100%, Figure 6d The relative changes are shown in .

[0199] <Example 3> CHO cell expression and analysis of DNP002

[0200] The humanized antibody DNP002 against CD66c was expressed and analyzed in CHO cells, which are used to express most therapeutic antibodies. To test the functional differences according to the subtype of the DNP002 antibody, IgG1 and IgG2 antibodies were prepared.

[0201] After codon optimization of the DNA sequences of the light chain variable region and the heavy chain variable region for the construction of humanized recombinant antibodies, they were synthesized and connected to the constant region of human IgG1 or IgG2 by overlapping PCR method, and the XhoI and EcoRI gene fragments were cloned into the pcDNA3.4 vector (Life Technology).

[0202] [Table 15]

[0203]

[0204]

[0205] In order to prepare the defucosylated DNP002 humanized antibody, when expressing the DNP002 IgG1 type antibody, 2F-PF (2F-peracetyl-fucose; Merck, catalog number: 344827) was added to the culture medium at 50uM and cultured, and then purified using a Mabselect sure protein A column (GE Healthcare Lifescience, catalog number: 11003494). The purified antibody was dialyzed with phosphate buffer, and the absorbance at 280nm was divided by an absorbance coefficient of 1.4 and converted to a concentration unit of "mg / mL", and then used in subsequent experiments.

[0206] Defucosylation was evaluated by comparing the reactivity of biotinylated lentil lectin (Vectorlaboratories, catalog number: B-1045) which has binding properties to fucose. Fucose-conjugated IgG1 DNP002 was reacted with biotinylated lentil lectin and used for TMB color development by SA-HRP (Jackson immunoresearch, catalog number: 016-030-084). However, the color development of defucosylated DNP002 was relatively less.

[0207] [Table 16]

[0208]

[0209] <Example 4> Study on the reactivity of DNP002 antibody with MDSC

[0210] The reactivity of DNP002 antibody against MDSCs was evaluated by flow cytometric analysis.

[0211] Specifically, after preparing blood from gastric cancer patients, DNP002 bound to APC and antibodies against MDSC marker antigens with different fluorescence (anti-HLA-DR-FITC, CD11b-PE, CD33-PE antibodies) were added to 100uL whole blood and reacted at 4°C for 20 minutes. 5ml of 1X RBC lysis buffer (ThermoFisher, catalog number: 00-4333-57) of red blood cell (RBC) lysis buffer was added to the product, reacted at room temperature for 30 minutes, centrifuged to remove the decomposed RBC, washed again with PBS, and performed by flow cytometry. The staining intensity is measured as the logarithm of the fluorescence intensity and expressed in tens.

[0212] In the result analysis, after specifying the monocyte area and granulocyte area excluding lymphocytes according to cell size in the dot plot, the group with no or low HLA-DR expression was selected, and the group with positive CD11b and CD33 was selected from these groups and designated as MDSC. The positive rate of DNP002 in the group designated as MDSC was confirmed ( Figure 7 ).

[0213] Specifically, in Figure 7From left to right in the figure above, 1) only monocytes and granulocytes other than lymphocytes in the FSC and SSC dot plots were gated. (FSC: forward scatter, a variable indicating the size of the cells to be analyzed, SSC: side scatter, a variable indicating the granularity of the cells to be analyzed and the degree of granules present in the cells), 2) HLA-DR low or (-) group was gated, and 3) MDSCs that were positive for CD11b and CD33 in the first and second groups. DNP002-positive MDSCs were 90.9% ( Figure 7 The upper right corner of the right dot plot), DNP002 negative MDSC is 4.4% ( Figure 7 (upper left in the right dot plot). MDSCs are divided into subtypes of monocytic MDSCs and granulocytic MDSCs. Granulocytic MDSCs express CD66b, but monocytic MDSCs do not, so the expression of CD66b can be used to distinguish MDSC subtypes.

[0214] Figure 7 The results of demonstrating the method of defining MDSC (HLA-DR low / (-) excluding lymphocytes, CD11b+, CD33+) and the reactivity to DNP002 (anti-CD66c) specific for MDSC. Since DNP002 binds to MDSC, DNP002 can be used as a method of specifying MDSC ( Figure 7 ), and DNP002 can induce ADCC effect to remove MDSCs ( Figure 8 ). In the following test results, the MDSCs bound to DNP002 were 90.9%.

[0215] Figure 8 The phenomenon that CD66b-positive granulocyte MDSCs were killed and their ratio was reduced by DNP002 treatment was shown. Figure 8 In the figure, the upper dot plot (control group) represents the sample before treatment with DNP002, while the lower dot plot (DNP002) is the result of MDSC reduction after treatment with DNP002. CD66b is a marker that can distinguish monocytic MDSC from granulocyte MDSC. In the following test results, most of the MDSCs are CD66b-positive granulocyte MDSCs, and DNP002 effectively kills granulocyte MDSCs.

[0216] As a result of analyzing the blood of 19 gastric cancer patients, the positive rate of DNP002 on MDSC in all PBMCs was 34.3-76.7%, and the average positive rate was 55.1%. Table 17 below shows the results of analyzing the reactivity of DNP002 antibody with MDSC using samples from 19 gastric cancer patients.

[0217] [Table 17]

[0218]

[0219]

[0220] <Example 5> The lysis effect of DNP002 on MDSC

[0221] 5.1.DNP002 lysis effect on whole blood MDSC

[0222] To examine the killing effect of DNP002 on MDSCs, 1×RBC lysis buffer (ThermoFisher, catalog number: 00-4333-57) was added to the blood of 5 gastric cancer patients to lyse the red blood cells, and then 1×10 5 DNP002 antibody was added to each well at a concentration of 10ug / mL and incubated in an incubator at 37°C for one day. After incubation, the cells were washed with PBS and reacted with antibodies against MDSC-marked antigens with different fluorescence (anti-HLA-DR, CD11b, CD33 antibodies) at 4°C for 20 minutes. After washing with PBS, flow cytometry was performed. The staining intensity was measured as the logarithm of the fluorescence intensity and expressed in tens.

[0223] As a result of the above experiments, Figure 8 Representative results are shown, in which the DNP002 antibody effectively induced apoptosis of MDSCs in the blood, and the ratio of MDSCs was significantly reduced compared with pre-treatment with the DNP002 antibody. Fig. 9 The results show that blood samples from 5 gastric cancer patients were Figure 8 Graph of the same test. The open bars represent the ratio of MDSCs before DNP002 treatment, while the solid bars represent the relative ratio of MDSCs after DNP002 treatment. After DNP002 treatment, it can be seen that the MDSC ratios of all 5 patient samples have been significantly reduced.

[0224] 5.2 Lysis effect of DNP002 on MDSCs in PBMCs

[0225] To more clearly elucidate the MDSC-targeted killing ability of the DNP002 antibody, only MDSCs were obtained under the exclusion of mature neutrophils, and the MDSC-killing effect of the DNP002 antibody was tested in the absence of the effect of neutrophils.

[0226] Specifically, Ficoll-Paque PLUS (Ge Healthcare, catalog number: 17-1440-02) solution was used to separate only the PBMC layer containing MDSCs from the blood of two gastric cancer patients. Blood cell density gradient separation using Ficoll solution effectively excludes mature neutrophils, so the killing effect of MDSCs can be analyzed more accurately. The prepared PBMCs were concentrated at 1×10 5 Each well was distributed into a 12-well plate, and the DNP002 antibody was added to each well at a concentration of 10 μg / mL, and then incubated at 37°C for 48 hours. At this time, the killing ability of MDSCs was compared using the antibody nivolumab (Bristol-Myers Squibb) against PD-1. After incubation, the cells were washed with PBS, and the increase or decrease of the MDSC group was analyzed by flow cytometry ( Fig.10 ).

[0227] As a result of flow cytometry, the DNP002-treated group showed an average of about 49% apoptosis compared to the group not treated with DNP002. On the other hand, nivolumab used as a control showed only about 24% MDSC killing effect. In the MDSCs isolated from the two patients, the killing effect of MDSC was the same. Therefore, this experiment confirmed that the effect of DNP002 antibody killing MDSC is significant.

[0228] The lysis effect of DNP002 on MDSC was confirmed for whole blood (Example 5.1) and PBMC (peripheral blood mononuclear cells; Example 5.2), respectively. In whole blood and PBMC, including NK cells of patients who can induce ADCC, CEACAM6-positive cells can be lysed by DNP002 with ADCC. However, in whole blood, neutrophils that are positive for the CEACAM6 target antigen are mixed with MDSC, and it is difficult to say that only MDSC is selectively lysed. In order to clarify the selective lysis of MDSC by DNP002, another experiment was performed on PBMCs in which neutrophils were removed by layer separation by centrifugation (Example 5.2). Therefore, it was confirmed that the lysis of MDSC by DNP002 was obvious.

[0229] <Example 6> The lysis effect of different antibody isotypes on MDSC

[0230] In order to test the ability of DNP002 antibody to kill MDSC targets, three types of DNP002 antibodies were prepared. Antibodies have different affinities to FcrRIII (CD16) expressed on NK cells depending on the isotype of the antibody, and antibody-dependent cell-mediated cytotoxicity (ADCC) is proportional to affinity. The affinity of the IgG2 isotype to FcrRIII is very low and does not have ADCC efficacy, while the affinity of the IgG1 isotype to FcrRIII is high and has excellent ADCC efficacy. It has been reported that the ADCC efficacy of antibodies depends not only on isotype, but also on the sugar chain structure connected to the 297th asparagine of the antibody. In particular, when fucose is absent from sugar chains, ADCC efficacy is enhanced (Shitara K. et al., J Immunol Methods. 2005 Nov 30; 306 (1-2) IgG subclass-independent improvement of antibody-dependent cellular cytotoxicity by fucose removal from Asn297-linked oligosaccharides).

[0231] In vitro tests were performed to test the ability of the DNP002 antibody to kill MDSC targets depending on its isotype and defucosylation. 1X RBC lysis buffer (ThermoFisher, catalog number: 00-4333-57) was added to the blood of 5 gastric cancer patients, and then 1×10 5 Each well was added with three antibodies such as DNP002 IgG1, DNP002 IgG2, and defucosylated IgG1 at a concentration of 10ug / mL and incubated in an incubator at 37°C for one day. After incubation, the cells were washed with PBS and reacted with antibodies against MDSC-marked antigens with different fluorescence (anti-HLA-DR, CD11b, CD33 antibodies) at 4°C for 20 minutes. After washing with PBS, flow cytometry was performed. The staining intensity was measured as the logarithm of the fluorescence intensity and expressed in tens.

[0232] It was observed that in all five gastric cancer patients who were tested, the MDSC killing effect increased in the order of IgG2, IgG1, and defucosylated IgG1 types ( Fig.11a and 11b). The difference in MDSC killing effect according to isotype and degree of defucosylation (fucose content less than 10%) can be considered by the difference in affinity of antibodies for FcrRIII, and the MDSC killing effect can be understood as the ADCC effect caused by NK cells.

[0233] like Fig.11a As shown, this is a representative result of the type of DNP002 formulation that confirms the ability to effectively remove MDSC. Compared with the above figure (control), most of the MDSC are retained in DNP002 IgG2, but the proportion of MDSC in DNP002 IgG1 is significantly reduced. This test confirmed that the killing effect of DNP002 IgG1 on MDSC is significantly high, while the killing effect of DNP002 IgG2 on MDSC is not significant. Since the ADCC effect of the IgG2 isotype is absent or very low compared to the IgG1 isotype, the killing effect of DNP002 IgG1 on MDSC is inferred by ADCC.

[0234] like Fig.11b As shown, blood samples from five patients with gastric cancer were tested for Fig. 9 The same test and drawing, and the horizontal axis in the figure represents each of the 5 gastric cancer patients (P#1, P#2, P#3, P#4, P#5). The killing effect of MDSC was observed in the order of control, IgG2, IgG1 and defucosylated IgG1. The IgG2 isotype has no MDSC killing effect or the MDSC killing effect is not significant due to the lack of ADCC function, but in IgG1 with ADCC function and IgG1-defucosylation in which ADCC function is enhanced by defucosylation, the MDSC killing effect is excellent.

[0235] <Example 7> Cancer cell killing effect of DNP002 combined with natural killer cells

[0236] In vitro tests were performed to test the combined effects of DNP002 antibody and natural killer (NK) cells. After PBMCs were separated from normal blood of three subjects using Ficoll-Paque PLUS (Ge Healthcare, catalog number: 17-1440-02) solution, only CD56-positive natural killer cells were isolated using CD56 microbeads (Miltenyi Biotec, catalog number: 130-050-401). Fig.12a and 12b , the horizontal axis represents three blood donors who were the source of natural killer cells.

[0237] Gastric cancer cell line A549 and pancreatic cancer cell line AsPC-1, which are positive for the target antigen CEACAM6 of DNP002, were inoculated with 1×10 4 into a 96-well plate and 2 × 10 5 The previously isolated natural killer cells were treated with 10 μg / mL of DNP002 antibody and then incubated at 37°C for 6 hours.

[0238] As a result of measuring cell viability using the EZ-cytox Enhanced Cell Viability Kit (Daeil Lab), it was confirmed that the combination of DNP002 antibody and natural killer cells enhanced the apoptotic effect in both cancer cell lines compared with single treatment ( Fig.12a and Figure 12b ).

[0239] This indicates that the cancer cell killing ability of DNP002 is significantly enhanced by the combination with natural killer cells. This indicates that the combination therapy with NK cells or NK cell therapeutic agents may be excellent for effectively removing CEACAM6-positive MDSCs and CEACAM6-positive cancer cells.

[0240] The selective lysis of MDSC by DNP002 in Example 5.2 and the combined effect with NK cells or NK cell therapeutic agents in Example 7 confirmed that the CEACAM6-positive cancer cells and CEACAM6-positive MDSC as targets can be eliminated. Although Examples 5.2 and 7 respectively show ADCC against different target cells (such as MDSC and cancer cells), in the case where both types of cells actually increase at the same time in cancer patients, DNP002 can remove both types of targets at the same time and can be used in combination with NK cell therapeutic agents to double the efficacy of simultaneously removing cancer cells and MDSC targets.

[0241] <Example 8> Detection of MDSCs in the cancer microenvironment of CEACAM6-negative patients

[0242] Since the CEACAM6 antigen is expressed not only in cancer cells but also in MDSCs, the use of the DNP002 antibody can detect not only cancer cells but also MDSCs. To test this, MDSCs but not cancer cells were detected in cancer patient tissues with positive or negative CEACAM6 antigen by immunohistochemistry.

[0243] Immunohistochemical staining was performed as follows. The tissue was dewaxed in xylene for 10 minutes, 3 times, in 100% alcohol for 10 minutes, 2 times, in 80% alcohol for 5 minutes, in 70% alcohol for 3 minutes, and then washed with triple distilled water. Then, peroxidase blocking was performed by immersion in 0.03% H2O2 for 10 minutes at room temperature and washed with triple distilled water. Immediately, the slide was placed in 1X citrate buffer (citrate buffer, pH 6.0), heated in boiling tap water for 20 minutes, slowly cooled, washed with triple distilled water, and then washed with 1X PBS. The 8F5 antibody, which is a monoclonal antibody of DNP002, was reacted at room temperature on the tissue area with 150ul (10ug / ml) per slide for 90±5 minutes. After the reaction, the slide was washed 4 times with 1X PBS, 5 minutes each time. The secondary antibody was reacted at room temperature for 20 minutes with 100ul per slide, and then washed 4 times with 1X PBST for 5 minutes each time. The DAB chromogen was developed at room temperature for 3 minutes with 100ul per slide, and the slides were washed with tap water for 10 minutes. Mayer's Hematoxylin was counterstained at room temperature for 3 minutes with 100ul per slide, and then washed under running tap water for 10 minutes. After dehydration, the slides were placed.

[0244] As a result of CEACAM6 immunostaining in lung adenocarcinomas, which are CEACAM6-positive in cancer cells, and lung squamous cell carcinomas, urinary bladder cancer, and malignant melanomas, which are CEACAM6-negative in the cancer cells themselves, it was confirmed that CEACAM6-positive MDSCs ( Fig.13 ). This suggests that MDSCs infiltrating around cancer tissues can be used as targets for diagnosis and treatment, regardless of the expression of CEACAM6 antigen in cancer tissues or in cancer cells themselves.

[0245] Regardless of the extent of CEACAM6 on the cell surface of cancer cells, MDSC tend to increase in cancer patients, which can detect and confirm MDSC infiltrating the tumor microenvironment, as shown in Example 8. When considered together with the results of Example 5.2 showing selective cleavage of MDSC, this suggests that MDSC can be used as a target for diagnostic and therapeutic purposes regardless of whether CEACAM6 is positive on cancer cells. The presence or absence of CEACAM6 expression on the cell surface of cancer cells can vary depending on the cancer type, but in any case, MDSC will increase in most cancer types. Therefore, this suggests that DNP002 can be used for diagnostic and therapeutic purposes in most cancers by targeting MDSC. <110> Kumho HT, Inc. <120> Use for preventing and treating diseases related to myeloid-derived suppressor cells <130> OPP20191779KR <150> KR 10-2018-0054977 <151> 2018-05-14 <150> KR 10-2019-0055950 <151> 2019-05-13 <160> 79 <170> KoPatentIn 3.0 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDR-H1 of mouse or chimeric anti-CD66c antibodies <400> 1 Ala Ser Gly Tyr Ser Phe Thr Asp Tyr Thr Met Asn 1 5 10 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR-H2 of mouse or chimeric anti-CD66c antibodies <400> 2 Leu Ile Asn Pro Phe His Gly Gly Thr Val Ser Asn Gln Arg Phe Lys 1 5 10 15 Val <210> 3 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> CDR-H3 of mouse or chimeric anti-CD66c antibodies <400> 3 Val Arg Gly Asp Pro Val Arg His Tyr Tyr Ala Leu Ala Tyr 1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR-L1 of mouse or chimeric anti-CD66c antibodies <400> 4 Gly Ala Ser Glu Asn Val Tyr Gly Thr Leu Asn 1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR-L2 of mouse or chimeric anti-CD66c antibodies <400> 5 Gly Ala Thr Asn Leu Ala Asp 1 5 <210> 6 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> CDR-L3 of mouse or chimeric anti-CD66c antibodies <400> 6 Val Ala Thr Tyr Tyr Cys Gln Asn Val Leu Ser Ala Pro Tyr Thr 1 5 10 15 <210> 7 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region of mouse or chimeric anti-CD66c antibody <400> 7 Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Phe His Gly Gly Thr Val Ser Asn Gln Arg Phe 50 55 60 Lys Val Lys Ala Thr Leu Thr Val Asp Val Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Leu Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Val Arg Gly Asp Pro Val Arg His Tyr Tyr Ala Leu Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 <210> 8 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region of mouse or chimeric anti-CD66c antibody <400> 8 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Val Tyr Gly Thr 20 25 30 Leu Asn Trp Tyr Gln Arg Lys Gln Gly Lys Ser Pro Gln Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Asp Gly Met Ser Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Gln Tyr Ser Leu Lys Ile Ser Ser Leu His Pro 65 70 75 80 Asp Asp Val Ala Thr Tyr Tyr Cys Gln Asn Val Leu Ser Ala Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Ile 100 105 <210> 9 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> CDR-H1 of humanized anti-CD66c antibody <400> 9 Ala Ser Gly Tyr Ser Phe Thr Asp Tyr Thr Met His 1 5 10 <210> 10 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR-H2 of humanized anti-CD66c antibody <400> 10 Leu Ile Asn Pro Phe Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe Lys 1 5 10 15 Gly <210> 11 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR-L1 of humanized anti-CD66c antibody <400> 11 Gly Ala Ser Glu Asn Val Tyr Gly Thr Leu Ala 1 5 10 <210> 12 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> CDR-L1 of humanized anti-CD66c antibody <400> 12 Arg Ala Ser Glu Asn Val Tyr Gly Thr Leu Asn 1 5 10 <210> 13 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> CDR-L3 of humanized anti-CD66c antibody <400> 13 Phe Ala Thr Tyr Tyr Cys Gln Asn Val Leu Ser Ala Pro Tyr Thr 1 5 10 15 <210> 14 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region of humanized anti-CD66c antibody (8F5-human-VH5) <400> 14 Phe His Met Ala Asn Val His Gln Val Gln Leu Val Gln Ser Gly Ala 1 5 10 15 Glu Val Lys Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser 20 25 30 Gly Tyr Ser Phe Thr Asp Tyr Thr Met Asn Trp Val Arg Gln Ala His 35 40 45 Gly Gln Asn Leu Glu Trp Ile Gly Leu Ile Asn Pro Phe His Gly Gly 50 55 60 Thr Val Ser Asn Gln Arg Phe Lys Val Lys Ala Thr Leu Thr Val Asp 65 70 75 80 Val Ser Thr Asn Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp 85 90 95 Asp Thr Ala Val Tyr Tyr Cys Val Arg Gly Asp Pro Val Arg His Tyr 100 105 110 Tyr Ala Leu Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 15 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region of humanized anti-CD66c antibody (8F5-human-VH6) <400> 15 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ala Pro Gly Gln Asn Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Phe His Gly Gly Thr Val Ser Asn Gln Arg Phe 50 55 60 Lys Val Lys Ala Thr Leu Thr Val Asp Val Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Gly Asp Pro Val Arg His Tyr Tyr Ala Leu Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 16 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region of humanized anti-CD66c antibody (8F5-human-VH7) <400> 16 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Thr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Phe His Gly Gly Thr Val Ser Asn Gln Arg Phe 50 55 60 Lys Val Lys Ala Thr Leu Thr Val Asp Val Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Gly Asp Pro Val Arg His Tyr Tyr Ala Leu Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 17 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region of humanized anti-CD66c antibody (8F5-human-VH10) <400> 17 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Thr Met Asn Trp Val Lys Gln Ala Pro Gly Gln Asn Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Phe His Gly Gly Thr Val Ser Asn Gln Arg Phe 50 55 60 Lys Val Lys Ala Thr Met Thr Val Asp Val Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Gly Asp Pro Val Arg His Tyr Tyr Ala Leu Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 18 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region of humanized anti-CD66c antibody (8F5-human-VH11) <400> 18 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Asp Tyr 20 25 30 Thr Met His Trp Val Lys Gln Ala Pro Gly Gln Asn Leu Glu Trp Ile 35 40 45 Gly Leu Ile Asn Pro Phe Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Asn Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Gly Asp Pro Val Arg His Tyr Tyr Ala Leu Ala Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 19 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region of humanized anti-CD66c antibody (8F5-human-VK5) <400> 19 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Val Tyr Gly Thr 20 25 30 Leu Ala Trp Tyr Gln Arg Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Asn Val Leu Ser Ala Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 20 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region of humanized anti-CD66c antibody (8F5-human-VK7) <400> 20 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gly Ala Ser Glu Asn Val Tyr Gly Thr 20 25 30 Leu Asn Trp Tyr Gln Arg Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Asn Val Leu Ser Ala Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> twenty one <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region of humanized anti-CD66c antibody (8F5-human-VK8) <400> twenty one Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Glu Asn Val Tyr Gly Thr 20 25 30 Leu Asn Trp Tyr Gln Arg Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Gly Ala Thr Asn Leu Ala Asp Gly Met Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Asn Val Leu Ser Ala Pro Tyr 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> twenty two <211> twenty three <212> PRT <213> Artificial Sequence <220> <223> Framework 1 in the heavy chain variable region of the chimeric anti-CD66c antibody <400> twenty two Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Ile Ser Cys Lys 20 <210> twenty three <211> twenty three <212> PRT <213> Artificial Sequence <220> <223> Framework 1 in the heavy chain variable region of a humanized anti-CD66c antibody (8F5-human-VH5) <400> twenty three Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys 20 <210> twenty four <211> twenty three <212> PRT <213> Artificial Sequence <220> <223> Framework 1 in the heavy chain variable region of the humanized anti-CD66c antibody (8F5-human-VH6) <400> twenty four Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Ile Ser Cys Lys 20 <210> 25 <211> twenty three <212> PRT <213> Artificial Sequence <220> <223> Framework 1 in the heavy chain variable region of the humanized anti-CD66c antibody (8F5-human-VH7) <400> 25 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Met Lys Ile Ser Cys Lys 20 <210> 26 <211> twenty three <212> PRT <213> Artificial Sequence <220> <223> Framework 1 in the heavy chain variable region of the humanized anti-CD66c antibody (8F5-human-VH10) <400> 26 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys 20 <210> 27 <211> twenty three <212> PRT <213> Artificial Sequence <220> <223> Framework 1 in the heavy chain variable region of the humanized anti-CD66c antibody (8F5-human-VH11) <400> 27 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys 20 <210> 28 <211> twenty three <212> PRT <213> Artificial Sequence <220> <223> Framework 1 in the light chain variable region of the chimeric anti-CD66c antibody <400> 28 Asp Ile Gln Met Thr Gln Ser Pro Ala Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Glu Thr Val Thr Ile Thr Cys 20 <210> 29 <211> twenty three <212> PRT <213> Artificial Sequence <220> <223> Framework 1 in the light chain variable region of a humanized anti-CD66c antibody (8F5-human-VK5) <400> 29 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20 <210> 30 <211> twenty three <212> PRT <213> Artificial Sequence <220> <223> Framework 1 in the light chain variable region of the humanized anti-CD66c antibody (8F5-human-VK7) <400> 30 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20 <210> 31 <211> twenty three <212> PRT <213> Artificial Sequence <220> <223> Framework 1 in the light chain variable region of the humanized anti-CD66c antibody (8F5-human-VK8) <400> 31 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys 20 <210> 32 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Framework 2 in the heavy chain variable region of the chimeric anti-CD66c antibody <400> 32 Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile Gly 1 5 10 <210> 33 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Framework 2 in the heavy chain variable region of a humanized anti-CD66c antibody (8F5-human-VH5) <400> 33 Trp Val Arg Gln Ala His Gly Gln Asn Leu Glu Trp Ile Gly 1 5 10 <210> 34 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Framework 2 in the heavy chain variable region of a humanized anti-CD66c antibody (8F5-human-VH6) <400> 34 Trp Val Lys Gln Ala Pro Gly Gln Asn Leu Glu Trp Ile Gly 1 5 10 <210> 35 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Framework 2 in the heavy chain variable region of the humanized anti-CD66c antibody (8F5-human-VH7) <400> 35 Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile Gly 1 5 10 <210> 36 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Framework 2 in the heavy chain variable region of the humanized anti-CD66c antibody (8F5-human-VH10) <400> 36 Trp Val Lys Gln Ala Pro Gly Gln Asn Leu Glu Trp Ile Gly 1 5 10 <210> 37 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> Framework 2 in the heavy chain variable region of the humanized anti-CD66c antibody (8F5-human-VH11) <400> 37 Trp Val Lys Gln Ala Pro Gly Gln Asn Leu Glu Trp Ile Gly 1 5 10 <210> 38 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Framework 2 in the light chain variable region of the chimeric anti-CD66c antibody <400> 38 Trp Tyr Gln Arg Lys Gln Gly Lys Ser Pro Gln Leu Leu Ile Tyr 1 5 10 15 <210> 39 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Framework 2 in the light chain variable region of a humanized anti-CD66c antibody (8F5-human-VK5) <400> 39 Trp Tyr Gln Arg Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 40 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Framework 2 in the light chain variable region of the humanized anti-CD66c antibody (8F5-human-VK6) <400> 40 Trp Tyr Gln Arg Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 41 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Framework 2 in the light chain variable region of a humanized anti-CD66c antibody (8F5-human-VK8) <400> 41 Trp Tyr Gln Arg Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr 1 5 10 15 <210> 42 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Framework 3 in the heavy chain variable region of the chimeric anti-CD66c antibody <400> 42 Asn Gln Arg Phe Lys Val Lys Ala Thr Leu Thr Val Asp Val Ser Ser 1 5 10 15 Asn Thr Ala Tyr Met Glu Leu Leu Ser Leu Thr Ser Asp Asp Ser Ala 20 25 30 Val Tyr Tyr Cys Val Arg 35 <210> 43 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Framework 3 in the heavy chain variable region of the humanized anti-CD66c antibody (8F5-human-VH5) <400> 43 Asn Gln Arg Phe Lys Val Lys Ala Thr Leu Thr Val Asp Val Ser Thr 1 5 10 15 Asn Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala 20 25 30 Val Tyr Tyr Cys Val Arg 35 <210> 44 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Framework 3 in the heavy chain variable region of the humanized anti-CD66c antibody (8F5-human-VH6) <400> 44 Asn Gln Arg Phe Lys Val Lys Ala Thr Leu Thr Val Asp Val Ser Thr 1 5 10 15 Asn Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala 20 25 30 Val Tyr Tyr Cys Val Arg 35 <210> 45 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Framework 3 in the heavy chain variable region of a humanized anti-CD66c antibody (8F5-human-VH7) <400> 45 Asn Gln Arg Phe Lys Val Lys Ala Thr Leu Thr Val Asp Val Ser Thr 1 5 10 15 Asn Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala 20 25 30 Val Tyr Tyr Cys Val Arg 35 <210> 46 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Framework 3 in the heavy chain variable region of the humanized anti-CD66c antibody (8F5-human-VH10) <400> 46 Asn Gln Arg Phe Lys Val Lys Ala Thr Met Thr Val Asp Val Ser Thr 1 5 10 15 Asn Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala 20 25 30 Val Tyr Tyr Cys Val Arg 35 <210> 47 <211> 38 <212> PRT <213> Artificial Sequence <220> <223> Framework 3 in the heavy chain variable region of the humanized anti-CD66c antibody (8F5-human-VH11) <400> 47 Ala Gln Lys Phe Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr 1 5 10 15 Asn Thr Ala Tyr Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala 20 25 30 Val Tyr Tyr Cys Val Arg 35 <210> 48 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Framework 3 in the light chain variable region of the chimeric anti-CD66c antibody <400> 48 Gly Met Ser Ser Arg Phe Ser Gly Ser Gly Ser Gly Arg Gln Tyr Ser 1 5 10 15 Leu Lys Ile Ser Ser Leu His Pro Asp Asp 20 25 <210> 49 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Framework 3 in the light chain variable region of the humanized anti-CD66c antibody (8F5-human-VK5) <400> 49 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Arg Gln Tyr Ser 1 5 10 15 Leu Lys Ile Ser Ser Leu His Pro Asp Asp 20 25 <210> 50 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Framework 3 in the light chain variable region of the humanized anti-CD66c antibody (8F5-human-VK7) <400> 50 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Tyr Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp 20 25 <210> 51 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Framework 3 in the light chain variable region of a humanized anti-CD66c antibody (8F5-human-VK8) <400> 51 Gly Met Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Tyr Thr 1 5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp 20 25 <210> 52 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Framework 4 in the light chain variable region of the chimeric anti-CD66c antibody <400> 52 Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 1 5 10 <210> 53 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Framework 4 in the heavy chain variable region of the humanized anti-CD66c antibody (8F5-human-VH5) <400> 53 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 54 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Framework 4 in the heavy chain variable region of the humanized anti-CD66c antibody (8F5-human-VH6) <400> 54 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 55 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Framework 4 in the heavy chain variable region of the humanized anti-CD66c antibody (8F5-human-VH7) <400> 55 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 56 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Framework 4 in the heavy chain variable region of the humanized anti-CD66c antibody (8F5-human-VH10) <400> 56 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 57 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Framework 4 in the heavy chain variable region of the humanized anti-CD66c antibody (8F5-human-VH11) <400> 57 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 1 5 10 <210> 58 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Framework 4 in the light chain variable region of the chimeric anti-CD66c antibody <400> 58 Phe Gly Gly Gly Thr Lys Leu Glu Ile Ile 1 5 10 <210> 59 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Framework 4 in the light chain variable region of the humanized anti-CD66c antibody (8F5-human-VK5) <400> 59 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> 60 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Framework 4 in the light chain variable region of the humanized anti-CD66c antibody (8F5-human-VK7) <400> 60 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> 61 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Framework 4 in the light chain variable region of a humanized anti-CD66c antibody (8F5-human-VK8) <400> 61 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 1 5 10 <210> 62 <211> 363 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding the heavy chain variable region of a chimeric anti-CD66c antibody <400> 62 gaggtccagc tgcaacagtc tggacctgaa ctggtgaagc ctggagcttc aatgaagata 60 tcctgcaagg cttctggtta ctcattcact gactacacca tgaactgggt gaagcagagc 120 catggaaaga accttgagtg gattggactt attaatcctt tccatggtgg tactgtctcc 180 aaccagaggt tcaaggtcaa ggccacatta actgtagaca agtcatccaa cacagcctac 240 atggagctcc tcagtctgac atctgacgac tctgcggtct attactgtgt aagaggtgac 300 ccggtccgcc attactatgc tttggcctac tggggtcagg gaacctcagt caccgtctcc 360 tca 363 <210> 63 <211> 217 <212> DNA <213> Artificial Sequence <220> <223> Polynucleotide encoding the light chain variable region of a chimeric anti-CD66c antibody <400> 63 gacatccaga tgactcagtc tccagcttca ctgtctgcat ctgtgggaga aactgtcacc 60 atcacatgtg gagcaagtga gaatgtttac ggtactttaa attggtatca gcggaaacag 120 ggaaaatctc ctcagctcct gatctatggt gcaaccaact tggcagatgg catgtcatcg 180 aggttcagtg gcagtggttc tggtagacag tattctc 217 <210> 64 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> 8F01 primer for the first fragment forward <400> 64 attactcgag gccaccatga a 21 <210> 65 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> 8F02 primer for the first fragment reverse <400> 65 agttgaagcg ctgctcacag tca 23 <210> 66 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> 8F03 primer for the second fragment forward <400> 66 gtgagcagcg cttcaactaa ggg 23 <210> 67 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> 3E04 primer for the first fragment reverse <400> 67 agtcgaattc tcatttccca ggagag 26 <210> 68 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> 8F04 primer for the first fragment reverse <400> 68 agttgaagca gaagacactg tca 23 <210> 69 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> 8F05 primer for the first fragment forward <400> 69 gtgtcttctg cttcaactaa ggg 23 <210> 70 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> 3E01 primer for the first fragment forward <400> 70 attackcgag gccaccatga agtggg 26 <210> 71 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> 8F06 primer for the first fragment reverse <400> 71 aacagtccgc ttgatctcca gct 23 <210> 72 <211> twenty three <212> DNA <213> Artificial Sequence <220> <223> 3EL02(2) primer for the second fragment forward <400> 72 gagatcaagc ggactgttgc tgc 23 <210> 73 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> The second fragment reverse primer 3E08 <400> 73 attagaattc tcagcactcg ccgcgg 26 <210> 74 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> IgG1 heavy chain constant region <400> 74 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 75 <211> 993 <212> DNA <213> Artificial Sequence <220> <223> IgG1 in the DNA sequence <400> 75 gcttcaacta agggaccaag cgtattccca cttgctccat ctagcaagag cactagcgga 60 ggaacagctg ctttggggtg tttggtaaag gattactttc ccgaacctgt taccgtgagc 120 tggaacagcg gggctttgac aagtggcgtt catacatttc ctgccgtttt gcaaagcagc 180 ggcttgtata gcttgagctc tgttgttacc gttccaagct catctctggg cacacaaaca 240 tacatctgca acgtgaacca caagccctca aacaccaagg tggacaagaa ggtggagcca 300 aagtcttgcg acaagaccca cacctgtcca ccttgtccag cccctgaact cctggggggc 360 ccttcagttt ttctctttcc tcctaaacct aaagatacac tcatgatcag tcggacccct 420 gaagttacct gtgtggtggt cgatgtgtct catgaagatc ctgaagtcaa gtttaactgg 480 tatgtggacg gcgtggaggt gcataatgcc aagaccaagc ctcgggagga gcaatataat 540 tctacctatc gcgtcgtctc tgtcctcacc gtcctgcatc aggactggct gaatggcaaa 600 gagtataagt gcaaagtcag taacaaagcc ctccccgccc ccatagagaa aaccattagt 660 aaagccaaag ggcagccccg cgagccccag gtctatacac tgccccccag tagagacgag 720 ctgacaaaga atcaggtgtc tctgacatgc ctggtgaaag gcttttatcc ctctgacatt 780 gccgtcgagt gggagtctaa tgggcagccc gagaataatt ataagacaac accccccgtg 840 ctggacagtg acggctcatt tttcctgtat tcaaaactga cagtggacaa aagtcggtgg 900 cagcaggga atgtgttttc atgcagtgtc atgcacgagg ccctccacaa tcactatacc 960 cagaaatctc tgagtctctc tcctgggaaa tga 993 <210> 76 <211> 326 <212> PRT <213> Artificial Sequence <220> <223> IgG2 heavy chain constant region <400> 76 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325 <210> 77 <211> 981 <212> DNA <213> Private Sequence(Artificial Sequence) <220> <223> IgG2 antibodies and anti-DNA antibodies <400> 77 gcttccacca agggcccatc cgtgttccct ctggccccat gttctaggtc tacatctgag agcaccgccg ccctcggctg tctggtgaag gattattcc ccgagcccgt gaccgtgtct tggaacagcg gagccctgac tagcggagtg cacaccttcc cagctgtgct gcagagctcc ggcctgtaca gcctctcttc tgtggtgacc gtgccctcta gcaacttcgg aacacagacc 240 300. 300. 300. 300. 300. 300. 300. 300. 300. 300. 300. 300 aagtgctgtg tggagtgcc tccatgtcct gccccacctg tggctggacc ttctgtgttt 360 ctgttccctc caaagccaaa ggataccctg atgatcagca gaactcctga ggtgacctgt 420 gtggtggtgg acgtgagcca cgaggatcct gaggtgcagt ttaactggta cgtggatggc 480 gtggaggtgc ataacgctaa gacaaagcct agggaggagc agtttaacag caccttcaga 540 gtggtgagcg tgctgaccgt ggtgcaccag gattggctga acggcaagga gtataagtgt 600 aaggtgtcta acaagggcct gccagcccct attgagaaga ccatcagtaa gaccaaggga 660 cagcctaggg agcctcaggt gtacaccctg cctccttcca gagaggagat gacaaagaac 720 caggtgagcc tgacctgtct ggtgaagggc ttctacccta gcgatatcgc cgtggagtgg 780 gagagcaacg gccagcctga gaacaactac aagaccaccc cacctatgct ggacagcgat 840 ggctctttct tcctgtactc taagctgacc gtggacaaga gcagatggca gcagggcaac 900 gtgttttctt gttctgtgat gcacgaggcc ctgcacaacc actacaccca gaagtctctg 960 tctctgtctc caggcaagtg a 981 <210> 78 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain constant region <400> 78 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 79 <211> 324 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of the light chain constant region <400> 79 cggactgttg ctgctccatc tgtttttata tttcctccca gcgacgagca gctgaaaagc 60 ggcactgcct ctgtggtgtg tctgctgaat aatttttacc cccgggaagc caaagtccag 120 tggaaggtgg ataatgccct ccagtctggg aacagtcagg aaagtgtgac agaacaggat 180 agtaaggact ctacttatag cctctcttct acactgactc tgtcaaaggc cgactatgag 240 aagcataaag tgtatgcctg cgaggtgaca catcagggcc tgagttcacc cgtgacaaaa 300 tcttttaacc gcggcgagtg ctga 324

Claims

1. An immunopotentiator comprising an antibody that specifically binds to CD66c (cluster of differentiation 66c), wherein the CD66c is expressed in myeloid-derived suppressor cells (MDSCs), in, The antibody is a defucosylated IgG1 type antibody and is humanized, and The antibody comprises the following complementarity determining regions (CDRs): (1) CDR-H1, which consists of the amino acid sequence of SEQ ID NO: 1, CDR-H2, which consists of the amino acid sequence of SEQ ID NO: 2, CDR-H3, which consists of the amino acid sequence of SEQ ID NO: 3, CDR-L1, which consists of the amino acid sequence of SEQ ID NO: 4, CDR-L2, which consists of the amino acid sequence of SEQ ID NO: 5, and CDR-L3, which consists of the amino acid sequence of SEQ ID NO: 13; (2) CDR-H1, which consists of the amino acid sequence of SEQ ID NO: 1, CDR-H2, which consists of the amino acid sequence of SEQ ID NO: 2, CDR-H3, which consists of the amino acid sequence of SEQ ID NO: 3, CDR-L1, which consists of the amino acid sequence of SEQ ID NO: 12, CDR-L2, which consists of the amino acid sequence of SEQ ID NO: 5, and CDR-L3, which consists of the amino acid sequence of SEQ ID NO: 13; (3) CDR-H1, which consists of the amino acid sequence of SEQ ID NO: 9, CDR-H2, which consists of the amino acid sequence of SEQ ID NO: 10, CDR-H3, which consists of the amino acid sequence of SEQ ID NO: 3, CDR-L1, which consists of the amino acid sequence of SEQ ID NO: 12, CDR-L2, which consists of the amino acid sequence of SEQ ID NO: 5, and CDR-L3 consisting of the amino acid sequence of SEQ ID NO: 13; or (4) CDR-H1, which consists of the amino acid sequence of SEQ ID NO: 1, CDR-H2, which consists of the amino acid sequence of SEQ ID NO: 2, CDR-H3, which consists of the amino acid sequence of SEQ ID NO: 3, CDR-L1, which consists of the amino acid sequence of SEQ ID NO: 11, CDR-L2, which consists of the amino acid sequence of SEQ ID NO: 5, and CDR-L3, which consists of the amino acid sequence of SEQ ID NO:

13.

2. The immunopotentiator according to claim 1, wherein The heavy chain variable region of the antibody comprises the following framework sequence: (1) V-FR1, which consists of the amino acid sequence of SEQ ID NO: 24; V-FR2, which consists of the amino acid sequence of SEQ ID NO: 34; V-FR3 consisting of the amino acid sequence of SEQ ID NO: 44; and V-FR4 consisting of the amino acid sequence of SEQ ID NO: 54; (2) V-FR1, which consists of the amino acid sequence of SEQ ID NO: 25; V-FR2, which consists of the amino acid sequence of SEQ ID NO: 35; V-FR3 consisting of the amino acid sequence of SEQ ID NO: 45; and V-FR4 consisting of the amino acid sequence of SEQ ID NO: 55; (3) V-FR1, which consists of the amino acid sequence of SEQ ID NO: 26; V-FR2, which consists of the amino acid sequence of SEQ ID NO: 36; V-FR3 consisting of the amino acid sequence of SEQ ID NO: 46; and V-FR4 consisting of the amino acid sequence of SEQ ID NO: 56; or (4) V-FR1, which consists of the amino acid sequence of SEQ ID NO: 27; V-FR2, which consists of the amino acid sequence of SEQ ID NO: 37; V-FR3 consisting of the amino acid sequence of SEQ ID NO: 47; and V-FR4 consisting of the amino acid sequence of SEQ ID NO:

57.

3. The immunopotentiator according to claim 1, wherein The light chain variable region of the antibody comprises the following framework sequence: (1) L-FR1, which consists of the amino acid sequence of SEQ ID NO: 29; L-FR2, which consists of the amino acid sequence of SEQ ID NO: 39; L-FR3 consisting of the amino acid sequence of SEQ ID NO: 49; and L-FR4, which consists of the amino acid sequence of SEQ ID NO: 59; (2) L-FR1, which consists of the amino acid sequence of SEQ ID NO: 30; L-FR2, which consists of the amino acid sequence of SEQ ID NO: 40; L-FR3 consisting of the amino acid sequence of SEQ ID NO: 50; and L-FR4 consisting of the amino acid sequence of SEQ ID NO: 60; or (3) L-FR1, which consists of the amino acid sequence of SEQ ID NO: 31; L-FR2, which consists of the amino acid sequence of SEQ ID NO: 41; L-FR3 consisting of the amino acid sequence of SEQ ID NO: 51; and L-FR4, which consists of the amino acid sequence of SEQ ID NO:

61.

4. The immunopotentiator according to claim 1, wherein The antibody comprises the following variable regions: (1) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 15; and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 21; (2) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 18; and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 21; (3) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 16; and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 19; (4) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 15; and A light chain variable region consisting of the amino acid sequence of SEQ ID NO: 20; or (5) a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 17; and The light chain variable region consists of the amino acid sequence of SEQ ID NO:

20.

5. A pharmaceutical composition for treating MDSC-related diseases, comprising the immunopotentiator according to any one of claims 1 to 4, wherein: The MDSC-related disease is gastric cancer or pancreatic cancer.

Images