Pharmaceutical composition for treating and / or preventing cancer

Abstract

A conjugate obtained by binding a benzodiazepine to an antibody or an antigen-binding fragment thereof having immunological reactivity with MCEMP1 protein, the conjugate being an antibody-drug complex (ADC) having a strong anti-tumor effect and being useful for the treatment and / or prevention of cancer, particularly cancer expressing MCEMP1 protein on the cell surface.

Description

Pharmaceutical composition for treating and / or preventing cancer

[0001] The present invention relates to a conjugate of an antibody against MCEMP1 protein or an antigen-binding fragment thereof with a benzodiazepine, and a pharmaceutical use thereof for the treatment and / or prevention of cancer.

[0002] Various antibody drugs targeting specific antigen proteins on cancer cells have been applied to cancer treatment as cancer therapeutic agents with few side effects due to their cancer specificity. For example, Mast Cell-Expressed Membrane Protein 1 (MCEMP1) is expressed on the cell surface of several types of cancer, and antibodies against this MCEMP1 protein are known to be promising pharmaceutical applications for the treatment and / or prevention of cancer (Patent Document 1).

[0003] In addition, some benzodiazepines are known to exhibit antitumor activity by recognizing specific DNA sequences and binding to the minor groove (DNA minor groove), causing DNA damage and inhibiting cell proliferation; specific examples include pyrrolobenzodiazepines (PBDs), indolinobenzodiazepines (IGNs), and pyridinobenzodiazepines (PDDs).

[0004] In recent years, studies have been conducted to enhance the efficacy of antibody drugs against cancer, and in particular, the development of antibody-drug conjugates (ADCs), which conjugate antibodies with drugs that have strong cell killing ability directly, has been actively pursued (Non-Patent Documents 1 and 2). There are few successful examples of ADCs using benzodiazepines, but ZYNLONTA is an ADC in which tesirine, a PBD derivative, is linked to an antibody against CD19. ＴＭ(loncastuximab tesirine-lpyl) has been approved for use in large B-cell lymphoma, and has achieved complete or partial responses of 48% or more in relapsed / refractory diffuse large B-cell lymphoma (DLBCL) (Non-Patent Document 3). Other drugs that have been developed include robalpituzumab tesirine, in which tesirine is linked to an antibody against DLL-3; vadastuximab talirine (SGN-CD33A), in which talirine is linked to an antibody against CD33; camidanlumab tesirine (Cami), in which tesirine is linked to an antibody against CD25; and SGN-CD70a, in which SGD-1882 is linked to an antibody against CD70a.

[0005] WO2017 / 170322

[0006] Lancet Oncol. 2016;17:e256-62Pharm Res. 2015 Nov;32(11):3526-40Ther Adv Hematol. 2022 Mar Vol. 13 1-10

[0007] The object of the present invention is to create an ADC that utilizes benzodiazepines and exhibits strong antitumor effects.

[0008] As a result of extensive research, the present inventors have discovered that a conjugate of an antibody against the MCEMP1 protein or its antigen-binding fragment with a benzodiazepine exhibits an extremely strong antitumor effect compared to an antibody against the MCEMP1 protein alone, and further that the antitumor effect of an ADC comprising an antibody against the MCEMP1 protein or its antigen-binding fragment conjugated with a benzodiazepine is significantly superior to the antitumor effect of ADCs currently on the market, thereby completing the present invention.

[0009] Specifically, the present invention has the following features (1) to (14).

[0010] (1) A conjugate comprising an antibody or an antigen-binding fragment thereof immunologically reactive with the MCEMP1 protein and a benzodiazepine.

[0011] (2) The conjugate according to (1), which has immunological reactivity with the extracellular domain portion of the MCEMP1 protein.

[0012] (3) The conjugate according to (1), wherein the MCEMP1 protein has an amino acid sequence represented by SEQ ID NO: 2, 4, 6 or 8, or an amino acid sequence having 80% or more sequence identity with said amino acid sequence.

[0013] (4) The conjugate according to (2), wherein the extracellular domain portion of the MCEMP1 protein has an amino acid sequence represented by SEQ ID NO: 10, 12, 14 or 16, or has 80% or more sequence identity with said amino acid sequence.

[0014] (5) The conjugate according to any one of (1) to (4), wherein the antibody is a monoclonal antibody or a polyclonal antibody.

[0015] (6) The conjugate according to any one of (1) to (5), wherein the antibody or antigen-binding fragment thereof and the benzodiazepine are bound via a linker.

[0016] (7) The conjugate according to any one of (1) to (6), wherein the benzodiazepine is pyrrolobenzodiazepine (PBD), indolinobenzodiazepine (IGN), pyridinobenzodiazepine (PDD), or isoquinolidinobenzodiazepine (IQB), or a derivative thereof.

[0017] (8) The benzodiazepine is selected from the group consisting of DSB-120, SJG-136 (SG2000), DC-81, DSB-120, SJG-136, SG2057, SG2202, SG2285, SGD-1882, SGD-1910, SG3199, SG3249, SG2219, IMGN779, IMGN632, (S)—N-(4-aminophenyl)-4-(4-(4-((2-methoxy-12-oxo-6a,7a,7b) ,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamide)phenyl)-1-methyl-1H-pyrrole-2-carboxamide, D211, D221, D231, GWL-78 or KMR-28-39, or a derivative thereof.

[0018] (9) The conjugate according to either (7) or (8), wherein the derivative is Tesirine (SG3249), Talirine (SGD-1910), SG3364, SG3227, SG3140 (MC-Phe-Lys-PAB-SG2057), SG3170, SG3203 (MC-Phe-Lys-PAB-SG2057), SG3231, SG3400, SG3376, DGN642, DGN549, FGX5-67, FGX-2-62, or FGX11-38.

[0019] (10) A pharmaceutical composition comprising the conjugate according to any one of (1) to (9) as an active ingredient.

[0020] (11) A pharmaceutical composition for treating and / or preventing cancer, comprising the conjugate according to any one of (1) to (9) as an active ingredient.

[0021] (12) The pharmaceutical composition according to (11), wherein the cancer is a cancer that expresses MCEMP1 protein on the cell surface.

[0022] (13) The pharmaceutical composition according to (11) or (12), wherein the cancer is selected from the group consisting of leukemia, myelodysplastic syndrome, sarcoma, thymoma, mast cell tumor, and perianal adenocarcinoma.

[0023] (14) A method for treating and / or preventing cancer, comprising administering to a subject a conjugate comprising an antibody or antigen-binding fragment thereof immunologically reactive with MCEMP1 protein and a benzodiazepine.

[0024] The conjugate of the present invention not only exhibits a significantly stronger antitumor effect than an antibody against the MCEMP1 protein alone, but also has a significantly better antitumor effect than existing commercially available ADCs. Therefore, the conjugate of the present invention can be used as an active ingredient in pharmaceuticals, particularly pharmaceuticals for the treatment and prevention of cancer.

[0025] The antibody or antigen-binding fragment thereof having immunological reactivity with the MCEMP1 protein used in the present invention (hereinafter collectively referred to as "anti-MCEMP1 antibody") is characterized by having immunological reactivity with the full-length MCEMP1 protein or a fragment thereof.

[0026] Furthermore, the MCEMP1 protein is a single-transmembrane protein expressed on the cell surface, and one preferred embodiment of the anti-MCEMP1 antibody is an antibody against the extracellular domain of the MCEMP1 protein, or an antigen-binding fragment thereof.

[0027] The efficacy of anti-MCEMP1 antibodies can be determined by evaluating in vivo the inhibition of cancer cell growth and the proportion of surviving individuals in animals into which cancer cells have been injected or in which cancer cells have been allowed to overgrow, or by evaluating in vitro the inhibition of cancer cell growth in cancer cells expressing the polypeptide, as described below, or by evaluating in vitro the death of cancer cells due to cytotoxic activity mediated by immune cells or complement, or the reduction in viable cells, or the impairment of cell function.

[0028] Similarly, the efficacy of the conjugate of anti-MCEMP1 antibody and benzodiazepine used in the present invention can be determined by in vivo evaluation of the effect of suppressing cancer cell growth in animals into which cancer cells have been transplanted or in which cancer cells have been allowed to overgrow, or by in vitro evaluation of cancer cell death, reduction in viable cells, or impairment of cell function in cancer cells expressing the polypeptide.

[0029] In the present invention, the term "conjugate" refers to a compound in which an antibody or an antigen-binding fragment thereof is covalently linked to a benzodiazepine. The bond between the antibody or the antigen-binding fragment thereof and the benzodiazepine may be via a linker.

[0030] The benzodiazepine forming the conjugate according to the present invention is known as one of the substances that is toxic to cancer cells by recognizing a specific DNA sequence and binding to the minor groove (DNA minor groove), thereby interfering with DNA synthesis and inhibiting cell proliferation.

[0031] Furthermore, subjects for cancer treatment and / or prevention in the present invention include mammals such as humans, pet animals, livestock, and sport animals, with humans being the preferred subject.

[0032] The following describes the anti-MCEMP1 antibody, benzodiazepine, conjugate of anti-MCEMP1 antibody and benzodiazepine, pharmaceutical composition using the conjugate, and method for treating and / or preventing cancer according to the present invention.

[0033] <Anti-MCEMP1 antibody> In the present invention, the anti-MCEMP1 antibody refers to an antibody or an antigen-binding fragment thereof that is immunologically reactive with the full-length MCEMP1 protein or a fragment thereof. Here, "immunological reactivity" refers to the property of the antibody or its antigen-binding fragment binding to the MCEMP1 protein or a partial polypeptide thereof in vivo.

[0034] The amino acid sequence represented by SEQ ID NO: 2 in the sequence listing disclosed in the present invention is the amino acid sequence isolated as human MCEMP1 protein, the amino acid sequence represented by SEQ ID NO: 4 is the amino acid sequence of its canine homologue (homolog), the amino acid sequence represented by SEQ ID NO: 6 is the amino acid sequence of its feline homologue, and the amino acid sequence represented by SEQ ID NO: 8 is the amino acid sequence of MCEMP1 isolated as its mouse homologue.

[0035] In the present invention, antibodies or antigen-binding fragments thereof that bind to the extracellular domain of the full-length MCEMP1 protein are preferably used. Specifically, examples include polypeptides containing the extracellular domain of the MCEMP1 protein, such as the amino acid sequences represented by SEQ ID NO: 10 (human), 12 (dog), 14 (cat), or 16 (mouse), fragments thereof (preferably consisting of 7 or more consecutive amino acids of these amino acid sequences), or amino acid sequences that share 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and particularly preferably 99% or more sequence identity with these polypeptides. The antibodies of the present invention include all antibodies that bind to these polypeptides and demonstrate cancer cell death, a reduction in viable cells, or a decrease in cellular function. Here, "% sequence identity" refers to the percentage (%) of identical amino acids (or bases) relative to the total number of amino acids (or bases) when the two sequences are aligned (aligned) to maximize similarity, with or without gaps.

[0036] The anti-MCEMP1 antibody used in the present invention may be a monoclonal or polyclonal antibody, and may belong to any class of immunoglobulin molecule, such as IgG, IgE, IgM, IgA, IgD, or IgY, or any subclass, such as IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2.

[0037] Polyclonal antibodies immunologically reactive with the MCEMP1 protein or fragments thereof (polyclonal anti-MCEMP1 antibodies) can be obtained by immunizing mice, human antibody-producing mice, rats, rabbits, chickens, etc. with, for example, the native MCEMP1 protein, a fusion protein with GST or the like, or a partial peptide thereof, and then obtaining serum from the resulting serum using ammonium sulfate precipitation, protein A, protein G, DEAE ion exchange columns, affinity columns to which the MCEMP1 protein or partial peptides are bound, or the like.

[0038] The nucleotide sequence and amino acid sequence of the MCEMP1 protein or its homologue used in the immunization can be obtained, for example, by accessing GenBank (NCBI, USA) and using algorithms such as BLAST and FASTA (Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90: 5873-5877, 1993; Altschul et al., Nucleic Acids Res. 25: 3389-3402, 1997). Methods for producing the MCEMP1 protein can be obtained by referring to WO2017 / 170322, and cells expressing the MCEMP1 protein can also be used.

[0039] A monoclonal antibody immunologically reactive with the MCEMP1 protein or a fragment thereof (monoclonal anti-MCEMP1 antibody) can be obtained, for example, by immunizing a mouse with the full-length MCEMP1 protein or a fragment thereof, or with cells expressing the MCEMP1 protein on their surface (e.g., leukemia cell U937), fusing spleen cells isolated from the mouse with myeloma cells, and selecting a clone producing a monoclonal anti-MCEMP1 antibody from the resulting fused cells (hybridoma). The antibody produced by the selected hybridoma can be obtained by a method similar to the method for purifying polyclonal antibodies described above.

[0040] The antibody used in the present invention may be a human antibody, a humanized antibody, a chimeric antibody, a non-human animal antibody, a genetically recombinant antibody, or a multispecific antibody (for example, a bispecific antibody).

[0041] Human antibodies can be obtained by sensitizing human lymphocytes infected with EB virus with a protein, protein-expressing cells, or a lysate thereof, fusing the sensitized lymphocytes with myeloma cells such as human U266 cells, and obtaining antibodies immunologically reactive with the MCEMP1 protein or a fragment thereof from the resulting fused cells.

[0042] A humanized antibody is a modified antibody, also known as a reshaped human antibody. Humanized antibodies are constructed by grafting the complementarity-determining regions (CDRs) of an antibody derived from an immunized animal onto the CDRs of a human antibody. Genetic recombination, a common technique for this purpose, is also well-known. Specifically, for example, a DNA sequence designed to link the CDRs of a mouse or rabbit antibody with the framework regions of a human antibody is synthesized by PCR from several oligonucleotides engineered to have overlapping ends. The resulting DNA is ligated to DNA encoding the constant regions of a human antibody, incorporated into an expression vector, and then introduced into a host for production (see European Patent No. 239400 and WO 96 / 02576). The framework regions of the human antibody linked via the CDRs are selected so that the CDRs form a good antigen-binding site. If necessary, amino acids in the framework regions of the variable regions of the antibody may be substituted so that the complementarity-determining regions of the reshaped human antibody form an appropriate antigen-binding site (Sato K. et al., Cancer Research 1993, 53:851-856). Alternatively, they may be substituted with framework regions derived from various human antibodies (see WO99 / 51743).

[0043] Antibodies are heteromeric glycoproteins that usually contain at least two heavy chains and two light chains. Antibodies are composed of two identical light chains and two identical heavy chains. Heavy chains have a heavy chain variable region at one end followed by several constant regions. Light chains have a light chain variable region at one end followed by several constant regions. The variable regions exhibit specific variable regions called complementarity-determining regions (CDRs) that confer binding specificity to the antibody. Portions of the variable regions that are relatively conserved are called framework regions (FRs). Complete heavy and light chain variable regions each contain four FRs connected by three CDRs (CDR1 to CDR3).

[0044] The sequences of the constant and variable regions of human-derived heavy and light chains are available from NCBI (USA: GenBank, UniGene, etc.). For example, reference can be made to the sequences of the human IgG1 heavy chain constant region under accession number J00228, the human IgG2 heavy chain constant region under accession number J00230, the human light chain κ constant region under accession numbers V00557, X64135, X64133, etc., and the human light chain λ constant region under accession numbers X64132, X64134, etc.

[0045] A chimeric antibody is an antibody produced by combining sequences derived from different animals, such as an antibody consisting of a heavy chain variable region and a light chain variable region of a mouse antibody and a heavy chain constant region and a light chain constant region of a human antibody. Chimeric antibodies can be produced using known methods, for example, by linking DNA encoding an antibody V region with DNA encoding a human antibody C region, incorporating the resultant into an expression vector, and introducing the vector into a host for production.

[0046] Non-human animal antibodies can be obtained by immunizing animals with a sensitizing antigen according to known methods. A typical method is to inject the sensitizing antigen intraperitoneally, intradermally, or subcutaneously into animals such as mice. When injecting the sensitizing antigen, the antigen is mixed with an appropriate amount of various adjuvants, such as Freund's complete adjuvant (CFA), and then administered to the animal multiple times. After immunizing an animal and confirming that the serum contains anti-MCEMP1 antibodies, the serum can be obtained and purified using ammonium sulfate precipitation, protein A, protein G, DEAE ion exchange columns, affinity columns coupled with MCEMP1 protein or partial peptides, or the like, as described above. Furthermore, monoclonal antibodies can be obtained from non-human animals by collecting immune cells from the immunized animal and fusing them with myeloma cells. The fusion of the immune cells with myeloma cells can be carried out according to known methods (see Kohler, G. and Milstein, C. Methods Enzymol. (1981) 73, 3-46).

[0047] A recombinant antibody can be obtained by cloning an antibody gene from a hybridoma, incorporating it into an appropriate vector, introducing it into a host, and producing it using recombinant technology (see Carl, A.K., Borrebaeck, James, W. Larrick, THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom by MACMILLAN PUBLISHERS LTD, 1990).

[0048] A multispecific antibody is an antibody that has multiple antigen-binding sites in one molecule and is specific to multiple antigens, and a preferred embodiment is a bispecific antibody that is specific to two types of antigens.

[0049] Specific examples of the antigen-binding fragment used in the present invention include Fab and F(ab'). ２ In addition to fragments such as the above, single chain antibodies (scFv) are also included.

[0050] When an antibody is digested with the protease papain, the disulfide bonds (hinge regions) connecting the H chains are cleaved and the antibody is separated into three fragments, of which the two N-terminal fragments are called the Fab region, and these fragments constitute Fab.

[0051] F(ab') ２ When an antibody is digested with the protease pepsin, the antibody is cleaved at the N-terminus, including the hinge region, and this N-terminal fragment is called F(ab') ２ The fragment is called the F(ab') region. ２ is.

[0052] A single-chain antibody (scFv) is a single-chain variable region fragment in which variable regions (Fv) consisting of VH and VL, the minimum unit required for an antibody to recognize an antigen, are linked by a peptide linker. Single-chain antibodies (scFv) are generally produced by expressing them in recombinant Escherichia coli or recombinant mammalian cells, followed by separation and purification using affinity chromatography or the like.

[0053] The anti-MCEMP1 antibody used to obtain the conjugate of the present invention may have amino acids in the variable region (e.g., FR) or constant region substituted with other amino acids. The amino acid substitutions are single or multiple, for example, fewer than 15, fewer than 10, 8 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, or 2 or fewer amino acids, preferably 1 to 9 amino acids. The substituted antibody should have the same or higher antigen-specific binding properties and antigen-binding affinity as the unsubstituted antibody, and should not cause rejection reactions when administered to humans.

[0054] The anti-MCEMP1 antibody used in the present invention is expected to have a stronger antitumor effect if it has a higher binding affinity with the MCEMP1 protein on the surface of cancer cells. ７ M －１ , at least 10 ８ M －１ , at least 5 × 10 ８ M －１, at least 10 ９ M －１ , at least 5 × 10 ９ M －１ , at least 10 １０ M －１ , at least 5 × 10 １０ M －１ , at least 10 １１ M －１ , at least 5 × 10 １１ M －１ , at least 10 １２ M －１ , or at least 10 １３ M －１ It is desirable that:

[0055] The binding ability of the anti-MCEMP1 antibody used in the present invention to effector cells can be improved by substituting one, two, or several amino acids in the heavy chain constant region of the antibody, or by removing fucose bound to N-acetylglucosamine in the N-glycoside-linked sugar chain bound to the heavy chain constant region. The above may be achieved by amino acid substitution alone, or may be a composition with an antibody bound to fucose.

[0056] Antibodies in which one, two, or several amino acids in the heavy chain constant region have been substituted can be produced by referring to, for example, WO2004 / 063351, WO2011 / 120135, U.S. Patent No. 8,388,955, WO2011 / 005481, U.S. Patent No. 6,737,056, and WO2005 / 063351.

[0057] An antibody from which fucose bound to N-acetylglucosamine in the N-glycoside-linked sugar chain in the heavy chain constant region has been removed, or a cell producing the antibody, can be prepared with reference to U.S. Patent No. 6,602,684, European Patent No. 1,914,244, or U.S. Patent No. 7,579,170. An antibody from which fucose bound to N-acetylglucosamine in the N-glycoside-linked sugar chain in the heavy chain constant region has been removed, or a composition of an antibody to which fucose has been bound, or a cell producing the antibody, can be prepared with reference to, for example, U.S. Patent No. 8,642,292.

[0058] The polyclonal anti-MCEMP1 antibody used in the present invention, the method for producing the antibody, the method for purifying it, and the method for producing the MCEMP1 protein used for immunization can be obtained by referring to WO2017 / 170322.

[0059] Specific examples of anti-MCEMP1 antibodies of the present invention include the following antibodies described in the aforementioned WO2017 / 170322:

[0060] An antibody or antigen-binding fragment thereof that is immunologically reactive with a partial polypeptide of an MCEMP1 protein having an amino acid sequence represented by SEQ ID NO: 2, 4, 6 or 8 or an amino acid sequence that has 80% or more (preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 99% or more) sequence identity with said amino acid sequence.

[0061] An antibody or antigen-binding fragment thereof that is immunologically reactive with a partial polypeptide of the extracellular domain of MCEMP1 protein having an amino acid sequence represented by SEQ ID NO: 10, 12, 14 or 16 or an amino acid sequence having 80% or more (preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 99% or more) sequence identity with said amino acid sequence.

[0062] In the examples described below, the above-mentioned polyclonal antibody against the region of the MCEMP1 protein that is expressed on the cell surface of cancer cells (extracellular region) was used to prepare a conjugate with benzodiazepine, a DNA damaging agent, and its strong antitumor effect was confirmed.

[0063] <Benzodiazepines> Some benzodiazepines are known to exhibit antitumor effects by recognizing specific DNA sequences and binding to the minor groove (DNA minor groove), causing DNA damage and inhibiting cell proliferation, and in the present invention, benzodiazepines having such antitumor activity are preferably used.

[0064] Specific examples of benzodiazepines having antitumor activity include pyrrolobenzodiazepines (PBDs), indolinobenzodiazepines (IGNs), pyridinobenzodiazepines (PDDs), isoquinolidinobenzodiazepines (IQBs), or derivatives thereof.

[0065] PBDs have an aromatic ring A (A ring), ring B (B ring), and pyrrolo ring C (C ring). In ring B, the N10-N11 positions are either imine (N=C), carbinolamine (NH-CH(OH)), or carbinolamine methyl ether (NH-CH(OMe)). PBDs can also take the form of C2-unsaturated PBD dimers, PBD dimers with C2 aryl substitutions, or polypyrrole-PBD. PBDs have the ability to recognize and bind to specific DNA sequences; the preferred sequence is 5'PuGPu3'.

[0066] Specific examples of PBDs include the antitumor antibiotic anthramycin and its analogs, including abbeymycin, chicamycin, mazethramycin, neothramycin A, meothramycin B, porothramicin, prothracarcin, sibanomycin (DC-102), sibiromycin, tomamycin, didehydroanhydroanthramicin, spadicomycin, and DC-81.

[0067] Specific examples of PBD include compounds described in "Mantaj, J et al. 2016 Angew. Chem. Int Ed. 55, 2-29; D. Antonow and D. Thurston, Chem. Rev. 2011", "Leanna et al. J. Med. Chem. 2020, 63, 9603-9622", "Jhon et al. Expert Opinion on Biological Therapy Vol21, 2021, 931-943", and "Julia et al. Angew Chem Int Ed Engl. 2017 Jan 9; 56 (2): 462-488".

[0068] Specific examples of the composite compound of PBD and a linker include those disclosed in WO1993 / 018045, WO2000 / 046228, WO2002 / 083682, WO2005 / 040170, WO2005 / 110423, WO2005 / 085251, WO2006 / 135687, WO2010 / 043880, WO2013 / 055990, WO2013 / 055993, WO2014 / 057072, WO2014 / 159981, WO2014 / 057120, WO2015 / 052532, WO2015 / 181559, WO2016 / 057072, WO2016 / 159981, WO2016 / 057120, WO2017 / 052532, WO2017 / 181559, WO2018 / 057072, WO2018 / 159981, WO2018 / 057120, WO2018 / 052532, WO2018 ...057072, WO2018 / 159981, WO2018 / 057120, WO2018 / 052532, WO2 O2017 / 020972, WO2017 / 059289, WO2017 / 137555, WO2017 / 137556, WO2 No. 017 / 186894, WO2018 / 031662, WO2018 / 069490, WO2018 / 091646, WO201 8 / 146188, WO2018 / 146189, WO2018 / 182344, WO2018 / 192944, WO2019 / No. 034764, WO2019 / 065964, WO2019 / 096788, WO2019 / 104289, WO2019 / 126 No. 691, WO2019 / 224340, WO2020 / 006722, WO2020 / 079229, WO2020 / 07923 No. 9, WO2020 / 100954, WO2020 / 141923, WO2020 / 152462, WO2020 / 196474 , WO2020 / 196712, WO2021 / 137646, WO2022 / 063853, WO2016 / 083468, W O2014 / 057073, WO2014 / 057113, WO2014 / 057114, WO2014 / 057115, WO20 No. 14 / 057117, WO2014 / 057118, WO2014 / 057119, WO2014 / 057120, WO2014 / 057122, U.S. Patent No. 4,316,900, WO2020 / 245283, WO2019 / 197602, WO2004 / 043 No. 963, WO2005 / 085260, WO2006 / 111759, WO2010 / 010347, WO2010 / 04387 No. 7, WO2011 / 128650, WO2011 / 130598, WO2011 / 130613, WO2011 / 130616,WO2013 / 041606, WO2013 / 053871, WO2013 / 053873, WO2013 / 055987, WO2013 / 053872, WO2013 / 164593 , WO2013 / 164592, WO2014 / 096368, WO2014 / 140862, WO2014 / 140174, WO2015 / 052322, WO2015 / 052532 , WO2015 / 052533, WO2015 / 052534, WO2015 / 052535, WO2016 / 038383, WO2016 / 037644, WO2016 / 044560, WO2017 / 137553, WO2017 / 129652, WO2017 / 223275, WO2018 / 146199, and WO2018 / 182341.

[0069] Examples of IGN and IGN-linker composite compounds include those described in WO2017 / 015495, WO2017 / 015496, WO2012 / 128868, WO2012 / 112687, WO2012 / 112708, WO2016 / 036801, WO2017 / 015502, WO2012 / 112708, WO2016 / 036801, WO2017 / 015502, WO2012 / 112708, WO2016 / 036801, WO2016 / 036801, WO2016 / 036802, WO2016 / 036803, WO2016 / 036804, WO2016 / 036805, WO2016 / 036806, WO2016 / 036807, WO2016 / 036808, WO2016 / 036809 ...9, WO201 Examples thereof include compounds described in WO2018 / 140435, WO2018 / 195245, WO2018 / 098258, WO2010 / 091150, WO2016 / 036804, WO2019 / 133652, WO2020 / 102051, WO2020 / 102053, and WO2020 / 205564.

[0070] Examples of PDD and PDD-linker composite compounds include the compounds described in WO2012 / 152915, WO2015 / 166289, WO2017 / 032983, WO2016 / 198869, WO2017 / 194960, WO2019 / 043417, WO2020 / 049286, WO2020 / 157491, and WO2022 / 023735.

[0071] Examples of IQB and conjugate compounds of IQB and a linker include compounds described in WO2016 / 149546, WO2018 / 071455, WO2018 / 053552, WO2017 / 011803, WO2017 / 091615, U.S. Patent No. 9,974,864, U.S. Patent No. 9,504,694, and U.S. Patent No. 10,350,218.

[0072] The benzodiazepines used in the present invention may be monomers or dimers. In the case of dimers, they may be homodimers or heterodimers with other antitumor compounds. The dimers may be dimers bonded to each other via the 8th, 7th, or 2nd carbon of the benzodiazepine.

[0073] Benzodiazepines preferably used in the present invention include DSB-120, SJG-136 (SG2000), DC-81, DSB-120, SJG-136, SG2057, SG2202, SG2285, SGD-1882, SGD-1910, SG3199, SG3249, SG2219, IMGN779, IMGN632, (S)—N-(4-aminophenyl)-4-(4-(4-(( 2-methoxy-12-oxo-6a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamide)phenyl)-1-methyl-1H-pyrrole-2-carboxamide, D211, D221, D231, GWL-78 or KMR-28-39, or a derivative thereof.

[0074] Furthermore, the derivative is preferably a derivative in which a linker is bound to the compound, and specific examples include Tesirine (SG3249), Talirine (SGD-1910), SG3364, SG3227, SG3140 (MC-Phe-Lys-PAB-SG2057), SG3170, SG3203 (MC-Phe-Lys-PAB-SG2057), SG3231, SG3400, SG3376, DGN642, DGN549, FGX5-67, FGX-2-62, and FGX11-38.

[0075] <Conjugate of anti-MCEMP1 antibody and benzodiazepine> In the present invention, the form of bond between the anti-MCEMP1 antibody and the benzodiazepine in the conjugate of the anti-MCEMP1 antibody and the benzodiazepine is not particularly limited as long as it is a form that can maintain anti-tumor activity against cancer, but a form of bond in which a linker structure is formed between the anti-MCEMP1 antibody and the benzodiazepine is preferred.

[0076] Here, the linker refers to a compound capable of binding an anti-MCEMP1 antibody to a benzodiazepine. Various known linkers may be used, or the activator structure may be appropriately chemically modified to allow direct binding.

[0077] The type of linker and the details of the binding method can be determined in accordance with known methods (see, for example, Greg T. Hermanson, Bioconjugate Techniques, Third Edition, WO2004 / 010957 and WO2014 / 012479).

[0078] In embodiments of the invention, reactive groups attached to the anti-MCEMP1 antibody, benzodiazepine and linker include:

[0079] Unless specifically chemically modified, reactive groups present in the amino acid sequence of antibodies or glycoproteins modified with amino acids include primary amines (ε-amino), carboxyls, thiols (sulfhydryls), carbonyls (ketones or aldehydes), and hydroxyls. Primary amines are found at the N-terminus of polypeptides and in the side chains of lysine residues. They are positively charged under physiological conditions and are usually located on the outside of proteins, allowing them to be used for conjugation without altering the protein structure. Carboxyls are found at the C-terminus of polypeptides and in the side chains of aspartic acid and glutamic acid. Sulfhydryls are found in the side chains of cysteine ​​and form disulfide bonds that maintain the protein's higher-order structure. Ketones or aldehydes can be generated in glycoproteins by oxidizing glycosyl groups with sodium metaperiodate.

[0080] The conjugates of the present invention are prepared by attaching the benzodiazepine to a linker attached to the reactive group on the antibody, by attaching the benzodiazepine to a linker attached to the benzodiazepine, or by attaching the benzodiazepine directly to the antibody.

[0081] Reactive groups attached to the linker and benzodiazepine include:

[0082] Reactive groups that can react with amines include N-hydroxysuccinimide (NHS) esters, imidoesters, pentafluorophenyl esters, hydroxymethylphosphine, isothiocyanates, isocyanates, acyl azides, N-hydroxyl esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryls, carbodiimides, and carboxylic acid anhydrides.

[0083] Carbodiimides, diazoalkanes, diazoacetyl compounds, carbonyldiimidazoles are reactive groups that can react with carboxyls and amines.

[0084] Reactive groups that can react with thiols include maleimide, haloacetamide, pyridyl disulfide, thiosulfone, vinyl sulfone, haloacetyl, aziridine, acryloyl, and aryl.

[0085] Hydrazides and alkoxyamines are reactive groups that can react with aldehydes.

[0086] Reactive groups that can react with hydroxyl include epoxy, oxirane, carbonyldiimidazole, N,N'-disuccinimidyl carbonate, N-hydroxysuccinimidyl chloroformate, and isocyanate.

[0087] Isocyanate is a reactive group that can react to hydroxyl.

[0088] Photoreactive reactive groups include diazirine, aryl azide, aryl, benzophenol, and diazo compounds.

[0089] Specific examples of the linker having the reactive group include the following:

[0090] Examples of linkers having the same reactive group terminal include linkers having N-hydroxysuccinimide ester as the reactive group (e.g., Disuccinimidyl Glutarate (DSG), Disuccinimidyl Suberate (DSS), Bis(sulfosuccinimidyl)Suberate (BS3), Tris-(Succinimidyl)Aminotriacetate (TSAT), PEGylated Bis(Sulfosuccinimidyl)Suberate (BS(PEG))). ５ , BS(PEG) ９ ), Dithiobis (Succinimidyl Propionate) (DSP), 3,3'-dithiobis (sulfosuccinimidyl propionate) (DTSSP), ethylene glycol bis(succinimidyl succinate) (EGS), Sulfo-ethylene glycol bis(succinimidyl succinate (Sulfo-EGS), Dimethyl adipimidate・2HCl (DMA), Dimethyl pimelimidate・2HCl (DMP), Dimethyl suberimidate・2HCl (DMS), Dimethyl3,3'-dithiobispropionimidate・2H Cl (DTBP), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), Disuccinimidyl tartrate (DST), Bis[2-(Succinimidooxycarbonyloxy)ethyl]Sulfone (BSOCOES), and linkers with maleimide reactive groups (e.g., Bismaleimidoethane (BMOE), 1,4-bismaleimidobutane (BMB), Bismaleimidohexane (BMH), Tris(2-maleimidothyl)amine (TMEA), 1,8-bismaleimido-(PEG)). ２ (BM (PEG) ２ ), 1,8-bismaleimido-(PEG) ３ (BM (PEG) ３), Dithiobismaleimidoethane (DTME) is used.

[0091] The main linkers having different reactive group ends include linkers having NHS ester and maleimide reactive groups (e.g., AMAS, BMPS, GMBS, Sulfo-MBS, MBS, Sulfo-MBS, SMCC, Sulfo-SMCC, EMCS, Sulfo-EMCS, SMPB, Sulfo-SMPB, SMPH, LC-SMCC, Sulfo-KMUS, SM(PEG) ２ , SM(PEG) ４ , SM(PEG) ６ , SM(PEG) ８ , SM(PEG) １２ , SM(PEG) ２４ ), linkers with NHS ester and pyridyldithiol as reactive groups (e.g., SPDP, LC-SPDP, Sulfo-LC-SPDP, SMPT, (PEG) ４ SPDP, PEG12-SPDP), linkers with NHS ester and haloacetyl as reactive groups (e.g., SIA, SBAP, SIAB, Sulfo-SIAB), linkers with NHS ester and aryl azide as reactive groups (e.g., ANB-NOS, Sulfo-SANPAH, ATFB), linkers with NHS ester and diazirine as reactive groups (e.g., SDA, Sulfo-SDA, LC-SDA, SDAD, Sulfo-SDAD), carbodiimide as a reactive group (e.g., DCC, EDC, EDAC, NHS, Sulfo-NHS), linkers in which maleimide and hydrazide are reactive groups (e.g., BMPH, EMCH, MPBH, KMUH), linkers in which pyridyldithiol and hydrazide are reactive groups (e.g., PDPH), linkers in which isocyanate and maleimide are reactive groups (e.g., PMPI), and linkers in which NHS ester and psoralen are reactive groups (e.g., SPB) are used.

[0092] Other linkers include polypeptide-containing linkers, such as Fmoc-Ala-Ala-Asn-PAB, Fmoc-Ala-Ala-Asn(Trt)-PAB, and Fmoc-PEG. ３-Ala-Ala-Asn(Trt)-PAB, Fmoc-PEG ４ -Ala-Ala-Asn(Trt)-PAB, Fmoc-Ala-Ala-Asn-PAB-PNP, Fmoc-Ala-Ala-Asn(Trt)-PAB-PNP, Fmoc-PEG ３ -Ala-Ala-Asn(Trt)-PAB-PNP, Azide-PEG ４ -Ala-Ala-Asn(Trt)-PAB-PNP, Mal-PEG ４ -Ala-Ala-Asn(Trt)-PAB-PNP, Fmoc-Val-Cit-PAB-OH, Val-Cit-PAB-OH, Fmoc-Val-Cit-PAB-PNP, MC-Val-Cit-PAB, MC-Val-Cit-PAB-PNP, Phe-Lys(Trt)-PAB, Fmoc-Phe-Lys(Trt)-PAB-PNP, Fmoc-Gly3-Val-Cit-PAB, Fmoc-Gly3-Val-Cit-PAB-PNP, Ala-Ala-Asn-PAB TFA salt, etc. are used.

[0093] Also, Bis-PEG-acid, PEG Acid (e.g., Acid-PEG-TEMPO, Amino-PEG-acid, Amino-PEG-CH ２ CO ２ H, Aminoxy-PEG-acid, Azido-PEG-acid, Carboxy-PEG-sulfonic acid, Fmoc-N-amido-PEG-acid, Fmoc-N-amido-PEG-CH ２ CO ２ H, Fmoc-aminoxy-PEG-acid, Hydroxy-PEG-acid, Hydroxy-PEG-CH ２ CO ２ H, m-PEG-acid, m-PEG-(CH ２ ) ３ -acid, Methoxytrityl-N-PEG-acid, N-methyl-N-(t-Boc)-PEG-acid, Propargyl-PEG-acid, Propargyl-PEG-CH ２ CO ２H, Propargyl-PEG-(CH ２ ), ３ -acid, t-Boc-N-amido-PEG-acid, t-Boc-N-amido-PEG-CH ２ CO ２ H, t-Boc-Aminoxy-PEG-acid, Acid-PEG-PFP ester, Miscellaneous PEG acid,), PEG PFP ester (e.g., Acid-PEG-PFP ester, Bis-PEG-PFP ester), Bis-PEG-NHS, PEG Aldehyde (e.g., m-PEG-aldehyde, m-PEG-benzaldehyde, Ald-PEG-acid, Ald-PEG-amine, Ald-PEG-azide, Ald-PEG-NH-Boc, Ald-PEG-NHS ester, Ald-PEG-TFP ester, Ald-PEG-t-butyl ester), PEG Tosylate (e.g., Azido-PEG-Tos, Hydroxy-PEG-Tos, m-PEG-Tos, t-Boc-Aminoxy-PEG-Tos, Trifluoroethyl-PEG-Tos, Tos-PEG-acid, Tos-PEG-CH ２ CO ２ H, Tos-PEG-alkyne, Tos-PEG-t-butyl ester, Tos-PEG-CH ２ CO ２ tBu, Tos-PEG-Tos, S-acetyl-PEG6-Tos, N-Tos-N-(t-butoxycarbonyl)-aminoxy-PEG ４-Tos, Ms-PEG-Ms, Ms-PEG-t-butyl ester, PEG-Ms, Propargyl-PEG-Ms), Boc-PEG (e.g., Amino-PEG-t-Boc-Hydrazide, Azido-PEG-t-Boc-Hydrazide, Boc-NH-PEG-NH-Boc, Bromoacetamido-PEG-Boc-amine, m-PEG-ONHBoc, Mal-Alkyl-t-Boc-amine, N-Boc-PEG-alcohol, N-Boc-PEG-bromide, N-methyl-N-(t-Boc)-PEG-acid, t-Boc-N-amido-PEG-acid, t-Boc-N-amido-PEG-CH ２ CO ２ H, t-Boc-N-Amid0-PEG-amine, t-Boc-N-amido-PEG-azide, t-Boc-N-amido-PEG-NHS ester, t-Boc-N-amido-PEG-sulfonic acid), PEG NHS ester (e.g., Acid-PEG-NHS ester, Azido-PEG-NHS ester, Bis-PEG-NHS, Fmoc-PEG-NHS ester, m-PEG-NHS ester, m-PEG-NHS Carbonate, Mal-PEG-NHS ester, Propargyl-PEG-NHS ester, t-Boc-N-amido-PEG-NHS ester, t-Butoxycarbonyl-PEG-NHS ester), Fmoc-PEG (e.g., Fmoc-N-amido-PEG-acid, Fmoc-NH-PEG-CH ２ CO ２H, Fmoc-PEG-NHS ester), Biotin PEG (e.g., Biotin PEG-acid, Biotin PEG-alcohol, Biotin PEG-alkyne, Biotin PEG-amine, Biotin PEG-azide, Biotin PEG-DBCO, Biotin PEG-hydrazide, Biotin-PEG-Mal, Biotin-PEG-NHS, Biotin-EDA-PEG-NHS, Biotin-PEG-oxyamine, Biotin-PEG-PFP, Biotin-EDA-PEG-PFP, Biotin-PEG-Tetrazine, Biotin-PEG-TFP, Azide-SS-biotin, Biotin-PEG ３ -SS-azide, DBCO-S-S-PEG ３ -Biotin, Dde Biotin-PEG4-Alkyne, Dde Biotin-PEG ４ -Azide, Dde Biotin-PEG ４ -DBCO, Diazo Biotin-PEG ３ -Alkyne, Diazo Biotin-PEG ３ -Azide, Diazo Biotin-PEG ３ -DBCO, Diol Biotin-PEG ３ -Alkyne, Diol Biotin-PEG ３ -Azide, PC Biotin-PEG ３ -Alkyne, PC-Biotin-PEG ４ -PEG ４ -Alkyne, PC-Biotin-PEG ４ -PEG4-Alkyne, PC Biotin-PEG ３ -Azide, PC-Biotin-PEG4-PEG3-Azide, PC-Biotin-PEG ４ -NHS carbonate, PC DBCO-PEG ３ -Biotin, WSPC Biotin-PEG ３-DBCO, Fmoc-Lys (biotin-PEG)-OH, Fmoc-N-amido-(PEG-biotin)-acid, TAMRA-Azide-PEG-Biotin), PEG Phosphonate, Aminooxy PEG (e.g., Aminooxy-PEG-acid, Aminooxy-PEG-alcohol, Aminooxy-PEG-azide, Aminooxy-PEG-bromide, Aminooxy-PEG-methane, Aminooxy-PEG-Propargyl, Aminooxy-PEG-t-butyl ester, Aminooxy-PEG-Thiol, Bis-(Aminooxy)-PEG, t-Boc-Aminooxy-PEG-acid, t-Boc-Aminooxy-PEG-alcohol, t-Boc-Aminooxy-PEG-amine, t-Boc-Aminooxy-PEG-Azide, t-Boc-Aminooxy-PEG-Bromide, t-Boc-aminooxy-PEG-Methane, t-Boc-aminooxy-PEG-Propargyl, t-Boc-aminooxy-PEG-S-Ac, t-Boc-Aminooxy-PEG-Thiol, t-Boc-Aminooxy-PEG-Tos, Fmoc-aminooxy-PEG-acid, Trifluoroethyl-PEG-Aminooxy), Alkyne PEG (e.g., endo-BCN-PEG, exo-BCN-PEG, Propargyl-PEG-acid, Propargyl-PEG-CH ２ CO ２ H, Propargyl-PEG-(CH ２ ) ３ -acid, Propargyl-PEG-(CH ２ ) ３-methyl ester、Prropargyl-･Gmcrrylat e、Propargglll･･･alcohol、rrop argylmm･･mamine、.. G-methylamiine、| Propargylla. de、Propargyllm･･bromide、rro pargyl-･･omMaleimide、rropa gylmPEy-ｭs、Propargyllmm･om｢ｳester、PrropargylllEGsulffoiic I ester、Propargyl-･ym｣ｨ ２ 39 ２ tBu、Propargylll･･thiol、ｰcbd gummies carbonaate、 nooxy-EEorropargyl、 BismPropargyl-･1、mm PEGPropargyll) . ２ 39 ２ 2、2zido-E((3ｨ ２ ) ) ３ -methyl ester、｡zidomE･m｡crylatee｡ do-PEGalcoholl ２ ) ) ３ OH、｡zido-EGamine、｡zid o-PEmazide、｡zido-EMM I ylamine、｡zido-･mmethyll ester、｡zido-EE1mｮｳ esterr ２ 39 ２-NHS, Azido-PEG-oxazolidin-2-one, Azido-PEG-PFP ester, Azido-PEG-phosphonic acid, Azido-PEG-phosphonic acid ethyl ester, Azido-PEG-sulfonic acid, Azido-PEG-t-Boc-Hydrazide, Azido-PEG-t-butyl ester, Azido-PEG-CH ２ CO ２ -t-butyl ester, Azido-PEG-TFP ester, Azido-PEG-Tos, Aminooxy-PEG-azide, Bromo-PEG-azide, Bromoacetamido-PEG-azide, Carboxyrhodamine 110-PEG-Azide, Isothiocyanato-PEG-Azide, Isothiocyanato-PEG-Azide, m-PEG-azide, Propargyl-PEG-azide, TAMRA-PEG-Azide, t-Boc-N-Amino-PEG-Azide, t-Boc-Aminooxy-PEG-Azide, Thiol-PEG-Azide, Trifluoroethyl-PEG-Azide, Azido-PEG-amino acid, Azido-PEG ４ -4-nitrophenyl carbonate, S-Acetyl-PEG ３ -Azido, Azide, Trityl-PEG １０ -Azide), Alkyne PEG, DBCO-PEG, BCN-PEG, Propargyl-PEG, Bis-PEG-acid, Bis-PEG-NHS, Bis-PEG-PFP, Bis-Propargyl-PEG, Amine-PEG-Amine, Azido-PEG-azide, Bromo-PEG, Mal PEG are used.

[0094] Furthermore, Py-ds-Prp-Osu, Py-ds-dmBut-OSu, Py-ds-dmBut-OPFP, Py-ds-Prp-OPFP, MAL- HA-OSu, MAL-di-EG-OPFP, MAL-tri-EG-OPFP, MAL-tetra-EG-OPFP, N3-di-EG-OPFP, N3 -tri-EG-OPFP, N3-tetra-EG-OPFP, ALD-BZ-OSu, ALD-di-EG-OSu, ALD-tetra-EG-OSu, ALD-di-EG-OPFP, ALD-tetra-EG-OPFP, PHA-di-EG-OPFP, and PHA-tetra-EG-OPFP are used.

[0095] In another embodiment, polyethylene glycol (PEG) as described in WO2015 / 057699 and WO2017 / 165851 can be used to obtain a conjugate that is expected to have better in vivo kinetics.

[0096] In another embodiment, the linker described in US Pat. No. 10,808,039 or a polypeptide represented by -Gly-Gly-Phe-Gly- can be used.

[0097] The linker between the anti-MCEMP1 antibody and the benzodiazepine may be composed of a single type or multiple types.

[0098] Methods for preparing a conjugate of an anti-MCEMP1 antibody and a benzodiazepine include a method in which the benzodiazepine is conjugated using the ε-amino group of the lysine side chain of the antibody, and a method in which the benzodiazepine is conjugated using a thiol formed by reducing the cysteine ​​residues that form disulfide bonds in the antibody.

[0099] When using the ε-amino group of a lysine residue of an antibody, for example, an active ester (e.g., N-hydroxysuccinimide ester) is reacted to form an amide bond. In this case, since there are many lysine residues in an antibody, the binding reaction proceeds nonspecifically. In this embodiment, for example, sc-vc-PAB-pyrrolobenzodiazepine (PBD) or Osu-Glu-vc-PAB-pyrrolobenzodiazepine (PBD) can be used.

[0100] When using thiols forming disulfide bonds in the side chains of cysteine ​​residues of antibodies, a method is used in which the disulfide bonds on the antibody are converted to thiols using a reducing agent such as mercaptoethanol, followed by reaction with maleimide or α-haloamide. Furthermore, methods using sulfonephenyloxadiazole, 4-cyanoethynyloxy derivatives, etc., can be used to stabilize thiol-mediated bonds. These bonds are more stable for longer periods than bonds formed by the conjugation reaction of cysteine ​​to maleimide. Furthermore, since stability is improved when the imide ring formed by the thiol group attached to the maleimide is opened by hydrolysis to form an amide bond, a linker with an amino group near the imide group can also be used. Furthermore, the thiols of cysteine ​​residues in antibodies form disulfide bonds, and a benzodiazepine can be attached between them via two thiols. For example, cross-linking can be formed using a linker with two disulfide bond sites, which can be generated from an amide group with two sulfones at the β-position, or dibromomaleimide.

[0101] The conjugates of the present invention can be prepared, for example, using the THIOMAB™ technology or ThioBridge™, which are methods that allow a fixed number of thiol groups to be introduced into a specific portion of an antibody (see Nature Biotechnology 26, 925-932 (2008) or Biocojugate Chem., 25(6), 1124-1136 (2014)).

[0102] The conjugate of the present invention can be prepared, for example, by reducing an antibody with the reducing agent dithiothreitol (DTT) in a phosphate buffer solution to obtain an antibody having a thiol-reactive group, followed by formation of a conjugate with a benzodiazepine. In addition to the method using a reducing agent, the conjugate can also be obtained by adding a thiol group to the primary amine of a lysine residue in the antibody by introducing Traut's reagent (2-Iminothiolane or N-Succinimidyl S-Acetylthioacetate (SATA)).

[0103] The amount of thiol added to the antibody can be quantified, for example, by mixing a sample solution containing 5,5'-Dithiobis(2-nitrobenzoic acid) (DTNB) and an SH group with phosphate buffer (pH 8.0) and distilled water, adding a DTNB solution dissolved in phosphate buffer, Good's buffer, or Tris buffer, incubating for a certain period of time, and then measuring the absorbance at 412 nm (see G. L. Ellman, Arch. Biochem. Biophys., 82, 70 (1959)).

[0104] The thiol groups added by cleaving disulfide bonds in the antibody through reduction treatment are preferably subjected to a treatment (capping) to prevent re-formation of disulfide bonds, such as with N-ethylmaleimide (NEM) or 2-iodoacetamide (IAA).

[0105] Forming a conjugate by binding a benzodiazepine to a thiol group added to an antibody can be achieved by known methods. Specifically, for example, linker reagents having a maleimide group or a bromoacetamide group can be used as linker reagents that specifically bind to the thiol group of a reduced antibody. For example, N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC) is used as a linker having a maleimide group. In this case, the presence of an amino group in the benzodiazepine allows the formation of an amide bond with the N-succinimide group of SMCC, thereby obtaining a conjugate.

[0106] In another embodiment, an amide bond is first formed with SMCC at the amino group present in the activator, and then a thiol group added to the antibody side is reacted with the maleimide group of the benzodiazepine-bound SMCC to obtain a conjugate.

[0107] In another embodiment, a conjugate of an antibody and a benzodiazepine can be formed by using two linkers. For example, a conjugate can be prepared by forming an amide bond between a primary amino group present in a lysine residue on the antibody and the N-succinimide group of SATA (N-succinimidyl-S-acetylthioacetate), adding a thiol group to the antibody, and then synthesizing a benzodiazepine containing an amino group or one to which an amino group has been added according to a standard method, reacting it with SMCC to form an amide bond with the N-succinimide group in SMCC. A conjugate can then be obtained by reacting the maleimide group in the benzodiazepine-bound SMCC with the thiol group in the antibody-bound SATA.

[0108] In another embodiment, conjugates of antibodies and benzodiazepines can be prepared using, for example, maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl (mc-Val-Cit-PAB) as a linker. mc-val-Cit-PAB (mc-vc-PAB) is a linker that can be cleaved by intracellular proteases (e.g., cathepsin B). A thiol group is attached to an antibody dissolved in phosphate buffer using DTT or the like. Meanwhile, a benzodiazepine having an amino group is reacted with the benzyloxycarbonyl (PAB) in mc-Val-Cit-PAB to prepare a benzodiazepine bound to mc-val-Cit-PAB, which can then be reacted with the aforementioned thiol-attached antibody to obtain a conjugate.

[0109] In yet another embodiment, SATA is attached to a primary amino group in a lysine residue of an antibody to add a thiol group, while succinimidyl 3-(2-pyridyldithio)propionate (SPDP) is reacted with a benzodiazepine having an amino group to form an amide bond with the N-succinimide group in SPDP.

[0110] The linker used in the present invention is cleavable under intracellular conditions, liberating a substance with antitumor activity comprising a benzodiazepine and a portion of the linker within the cell. For example, it is a linker that is cleaved by intracellular peptidases or proteases. Preferred are linkers that are cleaved by lysosomal or endosomal proteases, cathepsin B, cathepsin D, or plasmin. Examples include linkers containing polypeptides (Val-Cit, Phe-Leu, or Gly-Phe-Leu-Gly) that can be cleaved by cathepsin B. More specifically, linkers described in U.S. Patent No. 6,214,345 can be used.

[0111] Furthermore, in another embodiment, as a means for improving the stability, solubility, and metabolism of the conjugate of the present invention in blood and the binding affinity of the benzodiazepine to the anti-MCEMP1 antibody, for example, a linker having glucuronic acid (preferably β-D-glucuronide) described in WO 2007 / 011968 can be used. Alternatively, means described in WO 2013 / 173337, WO 2015 / 095755, WO 2015 / 123679, and WO 2018 / 031690 can be used.

[0112] Furthermore, in another embodiment, benzodiazepines can be site-specifically bound to anti-MCEMP1 antibodies using the methods described in, for example, WO2006 / 65533 and WO2018 / 160683.

[0113] In yet another embodiment, a conjugate of an anti-MCEMP1 antibody conjugated with two or more drugs including a benzodiazepine can be obtained by the method described in WO 2018 / 112253. Preferably, one of the two or more drugs is a pyrrolobenzodiazepine (PBD).

[0114] To obtain a composition containing a conjugate of an anti-MCEMP1 antibody of the present invention and a benzodiazepine, the composition can be subjected to, for example, gel filtration chromatography, and then the peak with a higher molecular weight than the antibody before linker attachment can be isolated. To detect the mass of the conjugate while maintaining the intact bivalent antibody, for example, the method described in WO 2013 / 049410 can be used.

[0115] The number of benzodiazepines bound per antibody of the conjugate of the anti-MCEMP1 antibody and benzodiazepine of the present invention can be quantified according to known methods such as mass spectrometry, ELISA, electrophoresis, and chromatography such as HPLC.

[0116] <Anti-tumor effect of the conjugate> The conjugate of the anti-MCEMP1 antibody and benzodiazepine of the present invention has anti-tumor activity in vitro or in vivo. Anti-tumor activity means reduction, elimination, inhibition of growth, apoptosis, necrosis, or killing of targeted cancer cells. Therefore, the anti-tumor effect of the conjugate of the present invention can be determined by examining the anti-tumor activity against cancer.

[0117] The in vivo antitumor effect can be assessed by administering the conjugate to a cancer-bearing organism, measuring the size of the tumor after administration, and examining the size of the cancer over time. The antitumor effect of the present invention can also be assessed by examining the survival rate. It can also be assessed by examining the ability to produce cytokines or chemokines. The antitumor effect of the conjugate of the present invention can be further assessed by examining the prevention of cancer, metastasis, or recurrence.

[0118] The conjugate of the present invention is expected to have a stronger antitumor effect if it has a higher binding affinity with the MCEMP1 protein on the surface of cancer cells. ７ M －１ , at least 10 ８ M －１ , at least 5 × 10 ８ M －１ , at least 10 ９ M －１ , at least 5 × 10 ９ M －１ , at least 10 １０ M －１ , at least 5 × 10 １０ M －１ , at least 10 １１ M －１ , at least 5 × 10 １１ M －１ , at least 10 １２ M －１ , or at least 10 １３ M －１ It is desirable that:

[0119] The ability of the conjugate of the present invention to bind to MCEMP1 can be determined using binding assays such as surface plasmon resonance (SPR), ELISA, Western blotting, immunofluorescence, and flow cytometry.

[0120] As described above, the conjugates of the present invention have an enhanced anti-tumor effect compared to the anti-MCEMP1 antibody alone, and the enhancement rate is preferably 30% or more, more preferably 40% or more, even more preferably 50% or more, even more preferably 55% or more, even more preferably 60% or more, even more preferably 65% ​​or more, and most preferably 70% or more. The enhancement rate of the anti-tumor effect of the conjugates of the present invention compared to the anti-MCEMP1 antibody alone can be calculated by adding an effective amount of each to cancer cells under the same conditions and comparing the cell proliferation rates from day 3 onwards after the start of addition.

[0121] <Pharmaceutical Composition, Method for Treating and / or Preventing Cancer> The target of the pharmaceutical composition of the present invention for treating and / or preventing cancer is not particularly limited, as long as it is a cancer (cell) that expresses the MCEMP1 protein.

[0122] As used herein, the terms "tumor" and "cancer" refer to a malignant neoplasm and are used interchangeably.

[0123] The cancers targeted by the present invention may be any cancer that expresses the MCEMP1 protein on the cell surface. Furthermore, as shown in the examples below, the present invention exhibits unexpectedly superior antitumor effects to ADCs of antibodies targeting CD33 protein for cancers that express both the MCEMP1 protein and CD33 protein on the cell surface. Therefore, the present invention is a preferred option for treating and preventing cancers that express both the MCEMP1 protein and CD33 protein on the cell surface. Specifically, for cancer patients who express both the MCEMP1 protein and CD33 protein on the cell surface and who could be treated with an anti-CD33 antibody ADC, preferably MYLOTARG (registered trademark) (gemtuzumab ozogamicin), if the anti-CD33 antibody ADC is ineffective or cannot be administered due to side effects such as toxicity, the present invention is a preferred option for treating and preventing such cancer patients.

[0124] Specific examples of the cancer include leukemia, myelodysplastic syndrome, sarcoma, thymoma, mast cell tumor, and perianal adenocarcinoma. These specific cancers include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, leukocytic leukemia, and leukocytic leukemia. leukemia), basophilic leukemia, blastic leukemia, bovine leukemia, cutaneous leukemia, embryonic cell leukemia, eosinophilic leukemia, gross leukemia, leader cell leukemia leukemia, Schilling leukemia, stem cell leukemia, subleukemic leukemia, anaplastic cell leukemia, hairy cell leukemia, hemoblastic leukemia leukemia), hemocytoblastic leukemia leukemia), histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphocytic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphotropic leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelogranulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasma cell leukemia, promyelocytic leukemia, refractory anemia (RA), refractory anemia with sideroblasts (RARS), refractory anemia with excess blasts ( RAEB), accelerated RAEB (RAEB-T), preleukemia and chronic myelomonocytic leukemia (CMML), osteosarcoma, chordoma, chondrosarcoma, Ewing's sarcoma, malignant peripheral nerve sheath tumor, rhabdomyosarcoma, synovial sarcoma, angiosarcoma, cutaneous angiosarcoma, extraskeletal Ewing's sarcoma, GIST (gastrointestinal stromal tumor), liposarcoma, fibrosarcoma, infantile fibrosarcoma, myxofibrosarcoma, leiomyosarcoma, alveolar soft part sarcoma, undifferentiated pleomorphic sarcoma, clear cell sarcoma, dermatofibrosarcoma protuberans, epithelioid sarcoma, thymoma, mast cell tumor, perianal adenoma, perianal adenocarcinoma, and the like are included, but are not limited to these.

[0125] Furthermore, preferred subjects (patients) are mammals, including, for example, primates, pet animals, livestock, sport animals, etc., with humans, dogs, and cats being particularly preferred.

[0126] When the conjugates used in the present invention are used as pharmaceutical compositions, they can be formulated by methods known to those skilled in the art. For example, they can be used parenterally in the form of a sterile solution or suspension injection in water or other pharmaceutically acceptable liquid. For example, they can be formulated by appropriately combining them with pharmacologically acceptable carriers or vehicles, specifically, sterilized water, physiological saline, vegetable oils, emulsifiers, suspending agents, surfactants, stabilizers, flavoring agents, excipients, binders, etc., and mixing them in a unit dosage form required for generally accepted pharmaceutical practice. The amount of active ingredient in these formulations is such that an appropriate dose within the indicated range can be obtained.

[0127] When the conjugate of the present invention is used as a pharmaceutical composition, it can be formulated in a lyophilized state containing any salts, surfactants, buffers, sugars, and cryoprotectants (including some sugars).

[0128] Sterile compositions for injection can be formulated according to standard pharmaceutical practice using a vehicle such as distilled water for injection. Examples of aqueous solutions for injection include physiological saline, isotonic solutions containing glucose or other adjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodium chloride. These solutions may be used in combination with appropriate solubilizers, such as alcohols, specifically ethanol, polyalcohols such as propylene glycol and polyethylene glycol, and nonionic surfactants such as Polysorbate 80™ and HCO-60. Examples of oily solutions include sesame oil and soybean oil, which may be used in combination with solubilizers such as benzyl benzoate and benzyl alcohol. Furthermore, these solutions may be combined with buffers such as phosphate buffers and sodium acetate buffers, soothing agents such as procaine hydrochloride, stabilizers such as benzyl alcohol, phenol, and antioxidants. The prepared injection solutions are usually filled into appropriate ampoules.

[0129] Administration may be oral or parenteral, preferably parenteral, and specific examples include injections, intranasal administrations, pulmonary administrations, transdermal administrations, etc. Examples of injections include intravenous injections, intramuscular injections, intraperitoneal injections, subcutaneous injections, intratumoral injections, etc., which can be used for systemic or local administration.

[0130] Furthermore, an appropriate administration method can be selected depending on the patient's age, body weight, sex, symptoms, etc. The dosage of a pharmaceutical composition containing an antibody or a polynucleotide encoding an antibody can be selected, for example, from 0.0001 mg to 1000 mg per kg of body weight per administration, such as 0.5 mg, 1 mg, 2 mg, 3 mg, 5 mg, 10 mg, 20 mg, 50 mg, 75 mg, 100 mg, 200 mg, 500 mg, or 1000 mg per kg of body weight per administration. Alternatively, the dosage can be selected, for example, from the range of 0.001 to 100,000 mg / body per patient, although it is not necessarily limited to these values.

[0131] The dosage and administration method will vary depending on the patient's weight, age, sex, symptoms, etc., but can be appropriately selected by those skilled in the art.

[0132] By administering to a subject a pharmaceutical composition for treating and / or preventing cancer, which contains the conjugate of the present invention as an active ingredient, it is possible to treat and / or prevent the above-mentioned cancers that express MCEMP1 on the cell surface, preferably leukemia, myelodysplastic syndrome, sarcoma, thymoma, mast cell tumor, and perianal adenocarcinoma.

[0133] A pharmaceutical composition for treating and / or preventing cancer, which contains the conjugate of the present invention as an active ingredient, and one or more antitumor agents can be administered to a subject, either together or separately, in combination to treat and / or prevent cancer. Antitumor agents that can be used in the present invention include alkylating agents, platinum complexes, topoisomerase inhibitors, antimetabolites, anticancer antibiotics, alkaloid antitumor agents, immune checkpoint inhibitors, immunomodulators, antitumor agents that inhibit angiogenesis, and molecular targeted drugs, all of which are used in standard cancer treatments.

[0134] The present invention will be described in more detail below with reference to examples, but the scope of the present invention is not limited to these specific examples.

[0135] (Example 1) Preparation of human MCEMP1 protein (1) Construction of an expression vector for the extracellular domain of human MCEMP1 protein The DNA base sequence encoding the extracellular domain of human MCEMP1 protein was cloned based on SEQ ID NO: 9 by the following method.

[0136] PCR was performed using 1 ng of human MCEMP1 / pcDNA3.1 prepared in Example 1 of WO 2017 / 170322, 0.4 μM each of two primers (described in SEQ ID NOS: 17 and 18) containing NdeI and XhoI restriction enzyme cleavage sequences, 0.2 mM dNTP, and 1.25 U of PrimeSTAR HS DNA polymerase (Takara Bio), along with the accompanying buffer, to a total volume of 50 μl. Using a Thermal Cycler (BIO RAD), PCR was performed by repeating 30 cycles of 98°C for 10 seconds, 55°C for 15 seconds, and 72°C for 30 seconds. The two primers were used to amplify the region encoding SEQ ID NO: 10, which contains the amino acid sequence of the extracellular domain of the MCEMP1 protein. After PCR, the amplified DNA was electrophoresed on a 2% agarose gel, and a DNA fragment of approximately 0.3 kbp was purified using the Wizard SV Gel and PCR Clean-Up System (Promega). The amplified product obtained by the above PCR reaction and pET-30a(+) (ThermoFisher Scientific) were each digested with restriction enzymes NdeI and XhoI (both Takara Bio), and purified using the Wizard SV Gel and PCR Clean-Up System (Promega). The purified amplified product and pET30a(+) were ligated using Ligation High Ver. 2 (Toyobo) to obtain an expression vector encoding a human MCEMP1 extracellular domain / His fusion protein (hereinafter referred to as hMCEMP1ECD-His) (hereinafter referred to as pET-30a(+)-hMCEMP1ECD-His). Sequence analysis using a DNA sequencer confirmed that this was the DNA base sequence encoding hMCEMP1ECD-His. The sequence represented by SEQ ID NO: 19 represents the base sequence encoding hMCEMP1ECD-His, and the sequence represented by SEQ ID NO: 20 represents the amino acid sequence of hMCEMP1ECD-His.

[0137] (2) Preparation of hMCEMP1ECD-His The expression vector pET-30a(+)-hMCEMP1ECD-His was introduced into BL21(DE3) (Novagen) according to the attached protocol, and the plate was spread on a selective medium (LB plate containing 50 μg / ml kanamycin) and cultured. Colonies that grew on the selective medium were picked and inoculated into LB medium containing 25 μg / ml kanamycin, and the plate was shaken overnight at 37°C as a preculture. The precultured medium was added to LB medium containing 25 μg / ml kanamycin, and the plate was shaken at 37°C and cultured at 132 rpm. The absorbance was measured sequentially, and when the absorbance at 600 nm reached 0.6 to 0.7, isopropyl-β-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM to induce expression of hMCEMP1ECD-His, followed by shaking culture at 37°C for 3 hours.

[0138] The collected E. coli was suspended in a buffer containing 50 mM Tris-HCl (pH 8.0) and 300 mM NaCl, and then disrupted by sonication. The resulting centrifuged supernatant was applied to Ni Sepharose 6 Fast Flow (GE Healthcare Biosciences). After washing with a washing buffer containing 20 mM Tris-HCl, 300 mM NaCl, and 5 mM imidazole (pH 8.0), hMCEMP1ECD-His was eluted with an elution buffer containing 20 mM Tris-HCl, 300 mM NaCl, and 500 mM imidazole (pH 8.0). The eluted fraction was collected and replaced with PBS by dialysis. The dialyzed sample was purified by applying it to a gel filtration column HiLoad 16 / 60 Superdex 200 (GE Healthcare Biosciences) equilibrated with PBS, and the peak fraction was collected. After concentration using an Amicon Ultra MWCO 3k (Merck Millipore), sterile filtration was performed using an HT Tafflin Acrodisc 0.22 μm (PALL), and this was used in the following experiments.

[0139] Example 2: Preparation of polyclonal antibodies that bind to the extracellular domain of MCEMP1 protein (1) Preparation of polyclonal antibodies against hMCEMP1ECD-His To obtain antibodies that bind to the extracellular domain of human MCEMP1 protein, 1 mg of hMCEMP1ECD-His prepared in Example 1 was used as the antigen. The initial immunization sample was mixed with a complete Freund's adjuvant (CFA) solution, and the subsequent immunization samples were mixed with an incomplete Freund's adjuvant (IFA) solution. This solution was administered subcutaneously to rabbits five times every three weeks. Blood was then collected to obtain antisera containing polyclonal antibodies. This antisera was further purified by passing it through a protein G carrier column (GE Healthcare Biosciences) and substituted with PBS to obtain IgG-type polyclonal antibodies against hMCEMP1ECD-His (hereinafter referred to as "rabbit polyclonal anti-MCEMP1 antibodies").

[0140] (2) Analysis of the Immunological Reactivity of Rabbit Polyclonal Anti-MCEMP1 Antibody to MCEMP1 Protein: 100 μl of the 1 μg / ml hMCEMP1ECD-His protein solution prepared in Example 1 was added per well of a Maxisorp (ThermoFisher Scientific) plate and allowed to stand at 4°C for 18 hours. The solution was removed, and a PBS-T solution containing 3% skim milk was added and allowed to stand at room temperature for 3 hours. The solution was removed, and the rabbit polyclonal anti-MCEMP1 antibody obtained above was added and allowed to stand at room temperature for 2 hours. After washing each well three times with PBS-T, HRP-labeled anti-rabbit IgG (H+L) antibody (Invitrogen) diluted 5000-fold with PBS was added and allowed to stand at room temperature for 1 hour. After washing the wells three times with PBS-T, TMB substrate solution (ThermoFisher Scientific) was added and the wells were left to stand for 15 to 30 minutes for color development. After color development, the reaction was stopped by adding 1 N sulfuric acid, and the absorbance values ​​at 450 nm and 595 nm were measured using an absorption spectrometer. The absorbance value at 595 nm was subtracted from the absorbance value at 450 nm to calculate the color intensity. As a result, it was confirmed that the prepared rabbit polyclonal anti-MCEMP1 antibody is a polyclonal antibody that exhibits immunological reactivity with the MCEMP1 protein.

[0141] (3) Analysis of immunological reactivity of rabbit polyclonal anti-MCEMP1 antibody to MCEMP1 protein expressed on the cell surface. Next, we analyzed whether the rabbit polyclonal anti-MCEMP1 antibody prepared in (1) above exhibits specific reactivity to MCEMP1 protein expressed on the cell surface. 10 CHO-human MCEMP1 cells and 10 CHO-emp cells prepared in Example 3 of WO 2017 / 170322 were used. ６ The cells were centrifuged, and 0.1 μg (5 μl) of rabbit polyclonal anti-MCEMP1 antibody was added. The cells were then suspended in 95 μl of PBS containing 3% fetal bovine serum (FBS) and incubated on ice for 1 hour. After washing with PBS containing 3% fetal bovine serum (FBS), the cells were suspended in 0.2 μl of R-Phycoerythrin-conjugated goat anti-rabbit IgG antibody (ImmunoResearch) and 99.8 μl of PBS containing 3% fetal bovine serum (FBS) and incubated on ice for 1 hour. After washing with PBS containing 3% fetal bovine serum (FBS), the cells were measured for fluorescence intensity using a FACSVerse (Becton Dickinson). A control was prepared by the same procedure as above, using purified antibodies derived from normal rabbit serum (Fujifilm Wako Pure Chemical Industries) instead of the MCEMP1 polyclonal antibody. As a result, the rabbit polyclonal anti-MCEMP1 antibody showed higher fluorescence intensity in CHO-human MCEMP1 cells compared to the control, but did not react with CHO-emp cells, confirming that it is a polyclonal antibody that specifically reacts with the MCEMP1 protein expressed on the cell surface.

[0142] Example 3: Preparation of a conjugate of an anti-MCEMP1 antibody and a PBD derivative (SGD-1910) A conjugate of the rabbit polyclonal anti-MCEMP1 antibody prepared in Example 2 and SGD-1910 (CAS #1342820-51-2) was prepared according to a standard method. A purified rabbit polyclonal anti-MCEMP1 antibody was dissolved in PBS(-) at a concentration of 10 mg / ml. A PBS(-) solution containing 100 mM EDTA and 50 mM L-cysteine ​​was added to the polyclonal antibody solution at a ratio of 1:10, followed by a reduction reaction at 4°C for 1 hour. After reduction, the solution was replaced with PBS(-) containing 1 mM EDTA using a Zeba™ Spin Desalting Column (MWCO 70k or 40k) and allowed to stand at room temperature for 18 to 72 hours to reoxidize the polyclonal antibody. Propylene glycol was added to the reoxidized polyclonal antibody to a concentration of 50%. 10 mM SGD-1910 dissolved in DMSO was added to the polyclonal antibody solution at a molar ratio of antibody to SDG-1910 of 1:5, and the mixture was allowed to react at room temperature for at least 1 hour. After the reaction, SGD-1910 that did not bind to the antibody was removed using a Zeba™ Spin Desalting Column (MWCO 70k or 40k), and the solvent was replaced with PBS(-) (repeated twice). After replacing the buffer solution with PBS(-), the solution was filtered using a 0.22 μm sterilizing membrane filter to obtain a solution containing the rabbit polyclonal anti-MCEMP1 antibody-SDG-1910 (conjugate 1; DAR 2 to 4) of the present invention. Using the same procedure as above, a solution containing the rabbit control antibody described in Example 2, which does not react with the MCEMP1 protein, was also obtained in the same manner to obtain a solution containing the rabbit control antibody-SDG-1910 (control conjugate; DAR 2 to 4).

[0143] Example 4 Analysis of Immunological Reactivity of Conjugate 1 with MCEMP1 Protein 100 μl of 1 μg / ml hMCEMP1ECD-His protein solution prepared in Example 1 was added per well of a Maxisorp (ThermoFisher Scientific) and allowed to stand at 4°C for 18 hours. The solution was removed, and a PBS-T solution containing 3% skim milk was added, followed by allowing to stand at room temperature for 3 hours. The solution was removed, and the conjugate 1 obtained above was added, followed by allowing to stand at room temperature for 2 hours. After washing each well three times with PBS-T, HRP-labeled anti-rabbit IgG (H+L) antibody (Invitrogen) diluted 5000-fold with PBS was added, followed by allowing to stand at room temperature for 1 hour. After washing the wells three times with PBS-T, TMB substrate solution (ThermoFisher Scientific) was added and the wells were left to stand for 15 to 30 minutes for color development. After color development, the reaction was stopped by adding 1 N sulfuric acid, and the absorbance values ​​at 450 nm and 595 nm were measured using an absorption spectrometer. The absorbance value at 595 nm was subtracted from the absorbance value at 450 nm to calculate the color intensity. As a result, it was confirmed that the prepared conjugate 1 exhibited immunological reactivity with the MCEMP1 protein.

[0144] (Example 5) Analysis of immunological reactivity to MCEMP1 protein expressed on the cell surface Next, we analyzed whether conjugate 1 prepared in Example 3 exhibits specific reactivity to MCEMP1 expressed on the cell surface. 100 CHO-human MCEMP1 cells and 100 CHO-emp cells prepared in Example 3 of WO2017 / 170322 were used. ６The cells were centrifuged, and 0.1 μg (5 μl) of conjugate 1 was added. The cells were then suspended in 95 μl of PBS containing 3% fetal bovine serum (FBS) and allowed to stand on ice for 1 hour. After washing with PBS containing 3% fetal bovine serum (FBS), the cells were suspended in 0.2 μl of R-Phycoerythrin-labeled goat anti-rabbit IgG antibody (ImmunoResearch) and 99.8 μl of PBS containing 3% fetal bovine serum (FBS) and allowed to stand on ice for 1 hour. After washing with PBS containing 3% fetal bovine serum (FBS), the fluorescence intensity was measured using a FACSVerse (Becton Dickinson). A control conjugate that did not react with MCEMP1 was used as a control, following the same procedure as above, except that the antibody against MCEMP1 was replaced with a control conjugate that did not react with MCEMP1. As a result, it was confirmed that conjugate 1 showed higher fluorescence intensity in CHO-human MCEMP1 cells than in the control, but did not react with CHO-emp cells, i.e., it reacted specifically with the MCEMP1 protein expressed on the cell surface.

[0145] (Example 6) Analysis of Expression of MCEMP1 Protein and CD33 Protein on Cancer Cell Surface Next, the human leukemia cell line U937 cells were analyzed to determine whether human MCEMP1 protein and human CD33 protein were expressed on the cell surface, and the expression levels were compared. ６The cells were centrifuged and reacted with FcR blocking reagent, Human (Miltenyi Biotec) according to the attached protocol to suppress nonspecific binding to Fc receptors. 0.1 μg (5 μl) of the anti-MCEMP1 polyclonal antibody prepared in Example 2 or 0.1 μg (5 μl) of a commercially available rabbit anti-CD33 antibody (abcam: ab245698) was added, and the cells were suspended in 95 μl of PBS containing 3% fetal bovine serum and allowed to stand on ice for 1 hour. After washing with PBS containing 3% fetal bovine serum (FBS), the cells were suspended in 0.2 μl of R-Phycoerythrin-labeled goat anti-rabbit IgG antibody (ImmunoResearch) and 99.8 μl of PBS containing 3% fetal bovine serum (FBS), and allowed to stand on ice for 1 hour. After washing with PBS containing 3% fetal bovine serum (FBS), fluorescence intensity was measured using an LSRFortessaX-20 (Becton Dickinson). Separately, a control was prepared by the same procedure as above, using a purified antibody derived from normal rabbit serum (Fujifilm Wako Pure Chemical Industries, Ltd.) that did not react with MCEMP1 protein or CD33 instead of the anti-MCEMP1 polyclonal antibody. As a result, U937 cells treated with anti-MCEMP1 polyclonal antibody showed a 16-fold increase in mean fluorescence intensity compared to the control, while U937 cells treated with a commercially available rabbit anti-CD33 antibody showed a 72-fold increase in mean fluorescence intensity compared to the control. Therefore, expression of MCEMP1 protein and CD33 protein on the cell surface of the human leukemia cell line U937 cells was confirmed. Furthermore, it was revealed that the expression level of CD33 protein on the cell surface of the human leukemia cell line was more than four-fold higher than that of MCEMP1 protein.

[0146] The rate of increase in mean fluorescence intensity (MFI value) was calculated using the following formula 1: Rate of increase in mean fluorescence intensity (fold) = (MFI value of cells reacted with anti-MCEMP1 polyclonal antibody or anti-CD33 antibody) / (MFI value of cells reacted with control antibody).

[0147] (Example 7) Antitumor activity of conjugate 1 against cancer cells The antitumor activity of conjugate 1 prepared using the rabbit polyclonal anti-MCEMP1 antibody prepared in Example 3 against cancer cells was evaluated in vitro. The antitumor activity against U937 cells, which were found to express MCEMP1 protein and CD33 protein on the cell surface in Example 6, was evaluated.

[0148] First, U937 cells were dispensed into a flat-bottom 96-well plate. The U937 cells were suspended in RPMI-1640 medium containing 10% fetal bovine serum to a concentration of 5,000 cells / well, and then added at 90 μl / well.

[0149] Conjugate 1, control conjugate, rabbit polyclonal anti-MCEMP1 antibody, and MYLOTARG (registered trademark) (gemtuzumab ozogamicin; DAR 2-3), a commercially available ADC for acute myeloid leukemia in which ozogamicin, a calicheamicin derivative, is linked to an antibody against the CD33 protein, were diluted with PBS to a final concentration of 0.08 μg / ml and added at 10 μl / well, followed by incubation in 5% CO ２ After incubation, 10 μl / well of Cell Counting Kit-8 (Dojindo Laboratories) was added, and the cells were incubated in 5% CO for 2 hours. ２ Color development was carried out in the presence of HCl at 37° C. Absorbance was measured using a plate reader Multiskan FC.

[0150] Based on the absorbance measurement results, the cell proliferation rate was calculated according to the following formula to evaluate antitumor activity: Cell proliferation rate (%) = (antibody-added culture - no cells) / (normal culture - no cells) x 100 Antibody-added culture: the average absorbance measured under conditions in which cells were cultured with the addition of antibodyNormal culture: the average absorbance measured under conditions in which PBS was added instead of the addition of antibodyNo cells: the average absorbance measured under conditions in which cells were incubated without the addition of cells.

[0151] As a result, Conjugate 1, a conjugate of SGD-1910 and a rabbit polyclonal anti-MCEMP1 antibody against the MCEMP1 protein described in the present invention, exhibited a greater reduction in cell proliferation rate, i.e., stronger antitumor activity, than the rabbit polyclonal anti-MCEMP1 antibody and the control conjugate. Specifically, in U937 cells, Conjugate 1 resulted in less than 60% survival of cancer cells, compared with 100% for the negative control (normal culture conditions). Furthermore, MYLOTARG (registered trademark), which was evaluated as a comparative control because U937 cells express CD33 protein on their cell surface, resulted in 90% survival of cancer cells, compared with 100% for the negative control (normal culture conditions).

[0152] The hydrazone linker of MYLOTARG (registered trademark) is more unstable than the Val-Ala linker of the conjugate of an anti-MCEMP1 antibody and SGD-1910, and the payload is more likely to be released, resulting in nonspecific cytotoxicity (Bioconjugate Chem. 2010, 21, 5-13). Furthermore, as shown in Example 6, the expression level of CD33 protein on the surface of cancer cells is significantly higher than that of MCEMP1 protein. For these reasons, it was predicted that MYLOTARG (registered trademark) would have significantly superior efficacy to Conjugate 1, a conjugate of an anti-MCEMP1 antibody and SGD-1910. However, contrary to expectations, Conjugate 1 actually exhibited significantly superior antitumor activity to MYLOTARG (registered trademark).

Claims

1. A conjugate comprising an antibody or an antigen-binding fragment thereof immunologically reactive with the MCEMP1 protein and a benzodiazepine bound thereto.

2. The conjugate according to claim 1, which is immunologically reactive with the extracellular domain portion of the MCEMP1 protein.

3. The conjugate of claim 1, wherein the MCEMP1 protein has the amino acid sequence represented by SEQ ID NO: 2, 4, 6 or 8, or an amino acid sequence having 80% or more sequence identity with said amino acid sequence.

4. The conjugate according to claim 2, wherein the extracellular domain portion of the MCEMP1 protein has the amino acid sequence represented by SEQ ID NO: 10, 12, 14 or 16, or has 80% or more sequence identity with said amino acid sequence.

5. The conjugate of claim 1, wherein the antibody is a monoclonal or polyclonal antibody.

6. The conjugate of claim 1, wherein the antibody or antigen-binding fragment thereof and the benzodiazepine are linked via a linker.

7. The conjugate of claim 1, wherein the benzodiazepine is a pyrrolobenzodiazepine (PBD), an indolinobenzodiazepine (IGN), a pyridinobenzodiazepine (PDD) or an isoquinolidinobenzodiazepine (IQB), or a derivative thereof.

8. The benzodiazepine is selected from the group consisting of DSB-120, SJG-136 (SG2000), DC-81, DSB-120, SJG-136, SG2057, SG2202, SG2285, SGD-1882, SGD-1910, SG3199, SG3249, SG2219, IMGN779, IMGN632, (S)-N-(4-aminophenyl)-4-(4-(4-((2-methoxy-12-oxo-6 2. The conjugate of claim 1, which is a,7,8,9,10,12-hexahydrobenzo[e]pyrido[1,2-a][1,4]diazepin-3-yl)oxy)butanamido)-1-methyl-1H-pyrrole-2-carboxamide)phenyl)-1-methyl-1H-pyrrole-2-carboxamide, D211, D221, D231, GWL-78 or KMR-28-39, or a derivative thereof.

9. The conjugate of claim 1, wherein the derivative is Tesirine (SG3249), Talirine (SGD-1910), SG3364, SG3227, SG3140 (MC-Phe-Lys-PAB-SG2057), SG3170, SG3203 (MC-Phe-Lys-PAB-SG2057), SG3231, SG3400, SG3376, DGN642, DGN549, FGX5-67, FGX-2-62, or FGX11-38.

10. A pharmaceutical composition comprising the conjugate according to any one of claims 1 to 9 as an active ingredient.

11. A pharmaceutical composition for treating and / or preventing cancer, comprising as an active ingredient the conjugate according to any one of claims 1 to 9.

12. The pharmaceutical composition according to claim 11, wherein the cancer is a cancer that expresses MCEMP1 protein on the cell surface.

13. The pharmaceutical composition of claim 12, wherein the cancer is selected from the group consisting of leukemia, myelodysplastic syndrome, sarcoma, thymoma, mast cell tumor, and perianal adenocarcinoma.

14. A method for treating and / or preventing cancer, comprising administering to a subject a conjugate comprising an antibody or antigen-binding fragment thereof immunologically reactive with MCEMP1 protein and a benzodiazepine.