Fucosylation-inhibiting compounds and methods of use thereof

Abstract

The disclosure generally provides fucosylation-inhibiting compounds and methods for their use and for producing antibodies with reduced fucosylation.

Description

[Technical field]

[0001] The disclosure generally provides compounds, and their uses, for inhibiting fucosylation of proteins, such as antibodies. [Background technology]

[0002] L-fucose, also called 6-deoxy-L-galactose or simply fucose, is a monosaccharide that is a component of some N- and O-linked glycans and glycolipids in animals. Fucose is typically added as a terminal modification of glycans, including those attached to blood group antigens, selectins, and antibodies. Protein fucosylation is thought to play a role in mammalian development, and aberrant protein fucosylation has been proposed to be associated with human disease, including upregulation in cancer and rheumatoid arthritis. Removal of fucosylation has been shown to improve antibody binding and activity. Thus, there is a need for compounds that can be used to inhibit fucosylation. The present embodiments meet this need, as well as others. Summary of the Invention

[0003] In some embodiments, a method for inhibiting fucosyltransferase is provided.In some embodiments, the method comprises contacting fucosyltransferase with a composition comprising purpurogallin, hexachlorophene, acriflavinum, baicalein, epicatechin monogallate, epicatechin-3-monogallate, epicatechin-3,5-digallate, theaflavin monogallate, tannic acid, methacycline, anthralin, mitoxantrone hydrochloride, hycanthone, esculidine lactate, aurintricarboxylic acid, carboplatin, cisplatin, primuletin, chrysin, diometin, suramin, hematin, gossypol, or any combination thereof.In some embodiments, the fucosyltransferase is present in the cell that produces antibody.

[0004] In some embodiments, a method for inhibiting protein fucosylation in a cell is provided. In some embodiments, the method includes contacting the cell with a composition comprising purpurogallin, hexachlorophene, acriflavinum, baicalein, epicatechin monogallate, epicatechin-3-monogallate, epicatechin-3,5-digallate, theaflavin monogallate, tannic acid, methacycline, anthralin, mitoxantrone hydrochloride, hycanthone, esculidine lactate, aurintricarboxylic acid, carboplatin, cisplatin, primuletin, chrysin, diometin, suramin, hematin, gossypol, or any combination thereof to inhibit the fucosylation of the protein.

[0005] In some embodiments, a method for producing an antibody with reduced fucosylation is provided. In some embodiments, the method includes contacting a cell producing the antibody with a composition comprising purpurogallin, hexachlorophene, acriflavinum, baicalein, epicatechin monogallate, epicatechin-3-monogallate, epicatechin-3,5-digallate, theaflavin monogallate, tannic acid, methacycline, anthralin, mitoxantrone hydrochloride, hycanthone, esculidine lactate, aurintricarboxylic acid, carboplatin, cisplatin, primuletin, chrysin, diometin, suramin, hematin, gossypol, or any combination thereof. In some embodiments, the cell is contacted with the composition in vitro or in vivo. In some embodiments, the antibody is secreted from the cell. In some embodiments, a compound is contacted with a fucosyltransferase prior to the secretion of the antibody.

[0006] In some embodiments, a method for producing an antibody with an altered glycosylation pattern is provided. In some embodiments, the method comprises contacting a cell producing the antibody with a composition comprising purpurogallin, hexachlorophene, acriflavinum, baicalein, epicatechin monogallate, epicatechin-3-monogallate, epicatechin-3,5-digallate, theaflavin monogallate, tannic acid, methacycline, anthralin, mitoxantrone hydrochloride, hycanthone, esculidine lactate, aurintricarboxylic acid, carboplatin, cisplatin, primuletin, chrysin, diometin, suramin, hematin, gossypol, or any combination thereof. In some embodiments, the cell is contacted with the composition in vitro or in vivo. In some embodiments, the antibody is secreted from the cell. In some embodiments, a compound is contacted with a fucosyltransferase prior to the secretion of the antibody. "Altered glycosylation pattern" with respect to a protein, such as an antibody produced according to any of the methods described herein, refers to a protein that has a different glycosylation pattern compared to the protein produced from the same cell that has not been contacted with a composition or compound described herein.

[0007] In some embodiments, a composition is provided comprising purpurogallin, hexachlorophene, acriflavinum, baicalein, epicatechin monogallate, epicatechin-3-monogallate, epicatechin-3,5-digallate, theaflavin monogallate, tannic acid, methacycline, anthralin, mitoxantrone hydrochloride, hycanthone, esculidine lactate, aurintricarboxylic acid, carboplatin, cisplatin, primuletin, chrysin, diometin, suramin, hematin, gossypol, or any combination thereof, and a cell.

[0008] In some embodiments, a composition is provided comprising purpurogallin, hexachlorophene, acriflavinum, baicalein, epicatechin monogallate, epicatechin-3-monogallate, epicatechin-3,5-digallate, theaflavin monogallate, tannic acid, methacycline, anthralin, mitoxantrone hydrochloride, hycanthone, esculidine lactate, aurintricarboxylic acid, carboplatin, cisplatin, primuletin, chrysin, diometin, suramin, hematin, gossypol, or any combination thereof, and an antibody.

[0009] In some embodiments, a cell-free medium is provided that includes purpurogallin, hexachlorophene, acriflavinum, baicalein, epicatechin monogallate, epicatechin-3-monogallate, epicatechin-3,5-digallate, theaflavin monogallate, tannic acid, methacycline, anthralin, mitoxantrone hydrochloride, hycanthone, esculidine lactate, aurintricarboxylic acid, carboplatin, cisplatin, primuletin, chrysin, diometin, suramin, hematin, gossypol, or any combination thereof. In some embodiments, the medium further includes an antibody. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 shows a graph of inhibition of fucosylation using Purpurogallin. [Diagram 2] FIG. 1 shows a graph of inhibition of fucosylation using hexachlorophene. [Diagram 3] FIG. 1 shows a graph of inhibition of fucosylation using Acriflavinum. [Figure 4] FIG. 1 shows a graph of inhibition of fucosylation using epicatechin monogallate. [Diagram 5] FIG. 1 shows a graph of inhibition of fucosylation using epicatechin-3-monogallate. [Figure 6] FIG. 1 shows a graph of inhibition of fucosylation using epicatechin-3,5-digallate. [Figure 7] FIG. 1 shows a graph of inhibition of fucosylation using theaflavin monogallate. [Figure 8] FIG. 1 shows a graph of inhibition of fucosylation using tannic acid. [Figure 9] FIG. 1 shows a graph of inhibition of fucosylation using methacycline. [Figure 10] FIG. 1 shows a graph of inhibition of fucosylation using mitoxantrone hydrochloride. [Figure 11] FIG. 1 shows a graph of inhibition of fucosylation using hycanthone. [Figure 12] FIG. 1 shows a graph of inhibition of fucosylation using esculidine lactate. [Figure 13] FIG. 1 shows a graph of inhibition of fucosylation using aurintricarboxylic acid. [Figure 14] FIG. 1 shows a graph of inhibition of fucosylation using carboplatin. [Figure 15] FIG. 1 shows a graph of inhibition of fucosylation using cisplatin. [Figure 16] FIG. 1 shows a graph of inhibition of fucosylation using diomethin. [Figure 17] FIG. 1 shows a graph of inhibition of fucosylation using suramin. [Figure 18] FIG. 1 shows a graph of inhibition of fucosylation using hematin. [Figure 19] FIG. 1 shows a graph of inhibition of fucosylation using gossypol. [Figure 20] FIG. 1 shows reduction of antibody fucosylation by epicatechin monogallate (ECG). [Figure 21] FIG. 1 shows reduction of antibody fucosylation by epicatechin-3-monogallate (ECGC). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] As used herein, unless otherwise indicated, the term "about" is intended to mean ±5% of the value it modifies. Thus, about 100 means 95 to 105.

[0012] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0013] As used herein, the terms "comprising" (and any form of "comprising" such as "comprise," "comprises," and "comprised"), "having" (and any form of "having" such as "have" and "has"), "including" (and any form of "including" such as "includes" and "including"), or "containing" (and any form of "containing" such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. Any composition or method using the term "comprising" should also be understood to describe such composition or method as a composition "consisting of," "consisting of," or "consisting essentially of" the recited components or elements.

[0014] The term "antibody" encompasses immunoglobulins or antibody molecules, including polyclonal antibodies, monoclonal antibodies, including murine, human, humanized, and chimeric monoclonal antibodies, as well as antibody fragments. In some embodiments, the antibody is a recombinant antibody. "Recombinant antibody" refers to an antibody expressed from cells that have been genetically engineered to produce a particular antibody. Methods for producing recombinant antibodies are known, and any such method can be used.

[0015] In some embodiments, an antibody refers to a polypeptide that exhibits binding specificity to a specific antigen or target molecule. An intact antibody is a heterotetrameric glycoprotein composed of two light chains and two heavy chains. Usually, each light chain is linked to a heavy chain by one covalent disulfide bond, but the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain contains a variable domain (V H ) followed by several constant domains. Each light chain has a variable domain (V L ) at its other end and a constant domain, with the light chain constant domain aligned with the first constant domain of the heavy chain and the light chain variable domain aligned with the variable domain of the heavy chain. The light chains of antibodies of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa and lambda, based on the amino acid sequence of their constant domains. In some embodiments, the antibodies are IgA, IgD, IgE, IgG, and IgM type antibodies. In some embodiments, the antibodies are IgA1, IgA2, IgG1, IgG2, IgG3, and IgG4 type antibodies.

[0016] In some embodiments, the antibody is an antibody fragment. The term "antibody fragment" refers to a portion of the intact antibody, typically the antigen-binding or variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, diabodies, single-chain antibody molecules, and multispecific antibodies that bind to multiple targets or different epitopes of the same target.

[0017] In some embodiments, the antibody is an antibody-drug conjugate. The term "antibody-drug conjugate" refers to an antibody or antibody fragment bound to the drug. Examples of antibody-drug conjugates include, but are not limited to, brentuximab vedotin, cantuzumab ravtansine, gemtuzumab ozogamicin, ibritumomab tiuxetan, polatuzumab vedotin, sacituzumab govitecan, trastuzumab duocarmazine, trastuzumab emtansine, and inotuzumab ozogamicin.

[0018] Polyclonal antibodies are heterogeneous populations of antibody molecules derived from the sera of animals immunized with an antigen. See, e.g., Kohler and Milstein, Nature 256:495 497 (1975); U.S. Patent No. 4,376,110; Ausubel et al., eds., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley Interscience, NY, (1987, 1992); Harlow and Lane ANTIBODIES: A Laboratory Manual Cold Spring Harbor Laboratory (1988); and Colligan et al., eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), the contents of which are incorporated herein in their entirety. Such antibodies may be of any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD, and any subclass thereof. The hybridoma producing the mAb of the invention can be cultivated in vitro, in situ, or in vivo. Production of high titer mAbs in vivo or in situ is currently the preferred method of production.

[0019] As used herein, the term "monoclonal antibody" (mAb) refers to an antibody (or antibody fragment) obtained from a population of substantially homogenous antibodies. A monoclonal antibody is usually directed against a single antigenic determinant. The modifier "monoclonal" indicates that the antibody is substantially homogeneous in character and does not require production of the antibody by any particular method. For example, murine mAbs can be made by the hybridoma method of Kohler et al., Nature 256:495-497 (1975). Chimeric mAbs, comprising light and heavy chain variable regions derived from a donor antibody (such as, but not limited to, a mouse) associated with light and heavy chain constant regions derived from an acceptor antibody (such as, but not limited to, another mammal, including, but not limited to, a human), can be prepared by the method disclosed in U.S. Pat. No. 4,816,567 or any other method. Humanized mAbs, having CDRs derived from a non-human donor immunoglobulin (such as, but not limited to, mouse) and the remaining immunoglobulin-derived portions of the molecule derived from one or more human immunoglobulins, optionally with altered framework support residues to maintain binding affinity, can be obtained by the techniques disclosed in Queen et al., Proc. Natl. Acad. Sci. (USA), 86:10029-10032 (1989) and Hodgson et al., Bio / Technology, 9:421 (1991). Methods for humanizing antibodies are conventional and are not critical to the methods described herein.

[0020] As used herein, the term "in combination with" means that the described agents can be administered to or contacted with a cell, animal, or target together in a mixture, simultaneously as single agents, or sequentially as single agents in any order.

[0021] As used herein, the term "contact" refers to bringing two components into close proximity to each other. In some embodiments, the components may be mixed together or simply placed in the same container. A composition may also be considered to be in contact with another component (e.g., a subject, a cell, etc.) when it is administered to that other component. Administration may be by injection, addition by pipette, and other conventional administration methods.

[0022] As used herein, the term "inhibit" with respect to enzyme activity means to decrease the activity of the enzyme by any measurable amount.

[0023] As used herein, the term "reduce" in reference to a protein modification means to reduce the amount of said modification by a measurable amount.

[0024] In some embodiments, the compounds described herein are salts of the compounds. In some embodiments, the salts are pharma- ceutically acceptable salts of the compounds. As used herein, the phrase "pharma-ceutically acceptable salt(s)" includes, but is not limited to, salts of acidic or basic groups. Compounds that are basic can form a wide variety of salts with a variety of inorganic and organic acids. Acids that can be used to prepare pharma-ceutically acceptable acid addition salts of such basic compounds include sulfate, thiosulfate, citrate, maleate, acetate, oxalate, hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, bisulfite, phosphate, acid phosphate, isonicotinate, borate, acetate, lactate, salicylate, citrate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucuronic acid, and the like. Non-toxic acid addition salts, i.e., acids that form salts with pharmacologically acceptable anions, include, but are not limited to, salts, saccharates, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, bicarbonates, malonates, mesylates, esylates, napsydisylate, tosylates, besylates, orthophosphates, trifluoroacetates, and pamoates (i.e., 1,1'-methylene-bis-(2-hydroxy-3-naphthalene)). Compounds that contain an amino moiety can form pharma-ceutically acceptable salts with various amino acids in addition to the acids mentioned above. Compounds that are acidic can form base salts with various pharmacologically acceptable cations. Examples of such salts include, but are not limited to, alkali metal or alkaline earth metal salts, particularly calcium, magnesium, ammonium, sodium, lithium, zinc, potassium, and iron salts. The present invention also encompasses quaternary ammonium salts of the compounds described herein when the compounds contain one or more tertiary amine moieties.

[0025] The term "antigen" refers to a target to which an antibody binds.

[0026] The term "purified" with respect to a protein or antibody refers to a protein or antibody that is substantially free of other materials associated with the molecule in its natural or cellular environment. For example, a purified protein is substantially free of cellular material or other proteins from the cell or tissue from which the protein is derived. The term refers to a sample in which the isolated protein is sufficiently pure to analyze it, or is at least 70%-80% (w / w) pure, at least 80%-90% (w / w) pure, 90-95% pure; and at least 95%, 96%, 97%, 98%, 99%, or 100% (w / w) pure. In some embodiments, the antibodies produced according to any of the embodiments described herein are further purified after production.

[0027] The embodiments provided herein provide compositions and methods that can be used, for example, to reduce fucosylation of proteins. "Reduced fucosylation" in the context of a protein generally refers to the reduction of the addition of fucose to glycans via α(1,2), α(1,3), α(1,4), and α(1,6) linkages. Fucosylation in the context of an antibody refers to the addition of fucose to N-acetylglucosamine ("GlcNAc") at the reducing end of the antibody's N-linked glycans ("fucosylation"). "Reduced fucosylation" in the context of an antibody refers to the reduction of fucose molecules attached to N-acetylglucosamine ("GlcNAc") at the reducing end of the antibody's N-linked glycans compared to an untreated antibody.

[0028] Antibody-dependent cellular cytotoxicity (ADCC) is a mechanism of cellular immunity whereby effector cells of the immune system actively lyse target cells bound to a specific antibody. ADCC is one of the mechanisms by which antibodies act as part of the immune response to limit and contain infections and diseases. It has been shown that monoclonal antibodies with reduced amounts of fucose in their glycosylation pattern exhibit significantly higher ADCC activity compared to normal fucosylated antibodies. Without wishing to be bound by any particular theory, the mechanism behind the improved ADCC of low-fucose or fucose-free antibodies appears to be mediated by the increased affinity of the modified Fc region of the antibody for FcγR on target cells. Thus, the compounds, compositions, and methods provided herein can provide antibodies with higher ADCC activity.

[0029] Certain compounds or their salts are referred to herein. Table 1 shows the chemical structures of these compounds. Also included are salts of these compounds, such as the pharma- ceutically acceptable salts of the compounds shown. In some embodiments, these salts are the HCl salts of these compounds.

[0030] [Table 1] [Table 2] [Table 3] [Table 4] [Table 5] [Table 6]

[0031] In some embodiments, provided herein is a composition comprising one or more of the above compounds. In some embodiments, the composition comprises one or more of the above compounds and a cell. In some embodiments, the cell is a eukaryotic or prokaryotic cell. In some embodiments, the cell is capable of producing a protein. In some embodiments, the cell is a recombinant genetically modified cell. A "recombinant genetically modified cell" is a cell that has been modified with nucleic acid material that is exogenous to the cell, such as by transfection, transduction, or infection. In some embodiments, the cell is stably transduced or transfected with a nucleic acid molecule that encodes an antibody. In some embodiments, the cell produces an antibody.

[0032] A variety of cells or cell types can be used. Non-limiting examples of cells that can be present in the compositions or used in any of the methods provided herein include CHO cells, NS0 cells, Sp2 / 0 cells, HEK293 cells, PER.C6 cells, etc.

[0033] In some embodiments, the composition further comprises a cell culture medium. In some embodiments, the composition comprises a selection agent. In some embodiments, the composition comprises one or more antibiotics. A selection agent is an agent, such as an antibiotic, that is used to select cells that have been transfected or transduced with a particular nucleic acid molecule. A variety of selection agents can be used.Examples include Geneticin™ (G418; (2R,3S,4R,5R,6S)-5-amino-6-[(1R,2S,3S,4R,6S)-4,6-diamino-3-[(2R,3R,4R,5R)-3,5-dihydroxy-5-methyl-4-methylaminooxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-2-(1-hydroxyethyl)oxane-3,4-diol), hygromycin b (O-6-amino-6-deoxy-L-glycero-D-galacto-heptopyranosylidene-(1-2-3)-O -β-D-talopyranosyl(1-5)-2-deoxy-N3-methyl-D-streptamine), neomycin, blasticidin (4-amino-1-[4-({(3S)-3-amino-5-[[amino(imino)methyl](methyl)amino]pentanoyl}amino)-2,3,4-trideoxy-β-D-erythro-hex-2-enopyranuronosyl]pyrimidin-2(1H)-one), mycophenolic acid ((E)-6-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1H-2-benzofuran-5-yl)-4-methyl Hex-4-enoic acid), Zeocin™ (2-({2-[2-{[(6-amino-2-{3-amino-1-[(2,3-diamino-3-oxopropyl)amino]-3-oxopropyl}-5-methyl-4-pyrimidinyl)carbonyl]amino}-3-[(5-{[1-({2-[4-({4-[(diaminomethylene)amino]butyl}carbamoyl)-4',5'-dihydro-2,4'-bi-1,3-thiazol-2'-yl]ethyl}amino)-3-hydroxy-1-oxo-2-butanyl]amino}-3-hydroxy-4

[0033] Examples of suitable hydroxylase inhibitors include, but are not limited to, N,N-dimethyl-3'-[(O-methyl-L-tyrosyl)amino]-4,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl}oxy)-3,5-dihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-4-ylcarbamate, and puromycin (3'-deoxy-N,N-dimethyl-3'-[(O-methyl-L-tyrosyl)amino]adenosine), or any combination thereof.

[0034] In some embodiments, a composition comprising one or more compounds provided herein and an antibody. In some embodiments, the antibody is an antibody secreted and / or produced by a cell. In some embodiments, the antibody is an antibody selected from the group consisting of adalimumab, alemtuzumab, alirocumab, atezolizumab, avelumab, belimumab, benralizumab, bevacizumab, bezlotoxumab, blinatumomab, brentuximab vedotin, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, carotuximab, certolizumab pegol, cetuximab, claudiximab, claudiximab, daclizumab, daratumumab, denosumab, depatuxizumab mafodotin, dinutuximab, durvalumab, elotuzumab, enfortumab vedotin, enoblitzumab, gemtuzumab ozogamicin, golimumab, guselkumab, ibalizumab, ibritumomab tiuxetan, ifavotuzumab, inebilizumab, infliximab, inotuzumab Buozogamicin, ipilimumab, isatuximab, margetuximab, mepolizumab, necitumumab, nimotuzumab, nivolumab, obiltoxaximab, obinutuzumab, ocrelizumab, ofatumumab, olaratumab, omalizumab, palivizumab, pembrolizumab, pertuzumab, polatuzumab vedotin, racotumomab, ramucirumab, ranibizumab, raxibacumab, rituximab, ximab, rovalpituzumab tesirine, sacituzumab, sacituzumab govitecan, sarilumab, secukinumab, siltuximab, sirukumab, teprotumumab, tildrakizumab, tocilizumab, tositumomab, trastuzumab, trastuzumab duocarmazine, trastuzumab emtansine, ublituximab, ustekinumab, vedolizumab, or their biosimilars.

[0035] In some embodiments, the compositions provided herein are cell-free or substantially cell-free. A composition is substantially cell-free when the number of cells present in the composition is less than 50,000, less than 40,000, less than 30,000, less than 20,000, less than 10,000, or less than 5,000. In some embodiments, the composition comprises a cell-free medium comprising one or more compounds or salts thereof as described herein. The cell-free medium may also comprise any other components as described herein.

[0036] In some embodiments, the antibody is an antibody capable of binding to ErbB proteins, such as ErbB1, also known as HER1 or EGFR, ErbB2, also known as HER2 or HER2 / neu, ErbB3, also known as HER3, and ErbB4, also known as HER4. In some embodiments, the antibody binds to transforming growth factor proteins, such as, but not limited to, TGFB1, TGFB2, TGFB3, and TGFB4. In some embodiments, the antibody binds to vascular endothelial growth factor proteins, such as, but not limited to, VEGFR1, Flt-1, VEGFR2, Flk-1 / KDR, VEGFR3, and Flt4. In some embodiments, the antibody binds to receptor activator of nuclear factor kappa beta (RANK), also known as TRANCE receptor or TNFRSFIIA. In some embodiments, the antibody binds to a protein related to the signaling lymphocyte activation molecule family (SLAMF) proteins, such as, but not limited to, SLAMF1, SLAMF2, SLAMF3, SLAMF4, SLAMF5, SLAMF6, SLAMF7, and SLAMF8. In some embodiments, the antibody binds to a platelet-derived growth factor receptor protein, such as, but not limited to, PDGFRα and PDGFRβ. In some embodiments, the antibody binds to a protein of the killer cell immunoglobulin-like receptor (KIR) family, such as, but not limited to, KIR3DL3, KIR3DP1, KIR3DL4, and KIR3DL2. In some embodiments, the antibody binds to a major histocompatibility complex class I chain-related protein (MIC), such as, but not limited to, MICA and MICB. In some embodiments, the antibody binds to a protein of the tumor necrosis factor (TNF) family, such as, but not limited to, LT alpha, LT beta, FASL, 4-IBBL, OX40L, and TNF-related apoptosis-inducing ligand (TRAIL).In some embodiments, the antibody binds to a protein of the death receptor (DR) family, such as, but not limited to, DR1, DR2, DR3, DR4, DR5, DR6, DR7, and DR8. In some embodiments, the antibody binds to a protein of the cytotoxic T lymphocyte antigen family, such as, but not limited to, CTLA4. In some embodiments, the antibody binds to a programmed death receptor, such as, but not limited to, PD1. In some embodiments, the antibody binds to a protein of the carcinoembryonic antigen family, such as, but not limited to, CD66a, CD66b, CD66c, CD66d, CD66e, and CD66f. In some embodiments, the antibody binds to a receptor of the T cell immunoglobulin and mucin domain family, such as, but not limited to, TIM1, TIM2, TIM3, and TIM4. In some embodiments, the antibody binds to lymphocyte activation genes (LAG), such as, but not limited to, FAG3. In some embodiments, the antibody binds to a group of differentiation antigens, including, but not limited to, CD2, CD3, CD19, CD20, CD22, CD25, CD27, CD30, CD33, CD38, CD39, CD52, CD70, CD73, CD94, CD134, CD137, CD252, and CD340. In some embodiments, the antibody binds to a tumor-associated carbohydrate antigen, including, but not limited to, mucin-associated GalNAc-O-ser / thr, also known as Tn, and Neu5Acα2-6GalNAcα-O-Ser / Thr, also known as sialyl Tn. In some embodiments, the antibody binds to a developmental stage-specific embryonic antigen, including, but not limited to, SSEA-1 (also known as Lewis X), SSEA-2, SSEA-3, and SSEA-4. In some embodiments, the antibody binds to a Thomsen-Freidenreich antigen, also known as Gal-Gal-NAc. In some embodiments, the antibody binds to a Lewis-related antigen, such as, but not limited to, Lewis Y, sialyl Lewis X, or sialyl Lewis A.In some embodiments, the antibody binds to carbohydrate structures of glycosphingolipids classified into the series, including, but not limited to, -Globo, -Isoglobo, -Ganglio, -Isoganglio, -Lacto, -Neolacto, LactoGanglio, -Muco, -Neogala, -Mollu, -Arthro, -Schisto, and -Spirometo. In some embodiments, the antibody binds to gangliosides, including, but not limited to, GD1, GD2, GD3, GM1, GM2, FucosylGm1, Neu5GcGm3, and polysialic acid.

[0037] In some embodiments, the composition may also include one or more buffers, stabilizers, emulsifiers, or any combination thereof. In some embodiments, the composition includes water.

[0038] In some embodiments, the composition comprises one or more cell-permeable compounds. In some embodiments, the cell-permeable compounds include hypotonic buffers; organic compounds such as methanol, ethanol, acetone, toluene, DMSO, and alkyltrimethylammonium bromides; detergents such as saponin, TritonX-100, Tween-20, Tween-80, digitonin, and sodium dodecyl sulfate (SDS); and pore-forming cytolysins, e.g., beta-hemolytic cytolysins such as streptolysin-O (SLO) and perfringolysin-O (PFO); or any combination thereof. In some embodiments, the composition comprises at least one cell permeabilizing agent selected from the group consisting of hypotonic buffer, methanol, ethanol, acetone, toluene, DMSO, alkyltrimethylammonium bromide, saponin, TritonX-100, Tween-20, Tween-80, digitonin, sodium dodecyl sulfate (SDS), beta-hemolytic cytolysin, streptolysin-O (SLO), perfringolysin-O (PFO), or any combination thereof.

[0039] Thus, in some embodiments, a method for inhibiting fucosyltransferase is provided.As used herein, "fucosyltransferase" refers to an enzyme that catalyzes the addition of fucose to a target, such as a protein.An example of fucosyltransferase includes, but is not limited to, α1,6 fucosyltransferase, which may also be referred to as FUT8.Thus, in some embodiments, the fucosyltransferase that is inhibited is FUT8. In some embodiments, the method includes contacting a fucosyltransferase with a composition comprising purpurogallin, hexachlorophene, acriflavinum, baicalein, epicatechin monogallate, epicatechin-3-monogallate, epicatechin-3,5-digallate, theaflavin monogallate, tannic acid, methacycline, anthralin, mitoxantrone hydrochloride, hycanthone, esculidine lactate, aurintricarboxylic acid, carboplatin, cisplatin, primuletin, chrysin, diometin, suramin, hematin, gossypol, or any combination thereof, or any salt thereof. The fucosyltransferase may be present in a cell or in a cell-free system.

[0040] A variety of cells can contain fucosyltransferase. Examples of cells include, but are not limited to, CHO cells, NS0 cells, Sp2 / 0 cells, HEK293 cells, or PER.C6 cells. These cells are non-limiting examples and other cell types and cell lines can be used.

[0041] In some embodiments, cells contacted with the compositions and compounds described herein produce antibodies. In some embodiments, the antibodies are recombinant antibodies. In some embodiments, the antibodies are directed to the following targets: IL8, ErbB, ErbB1, ErbB2, ErbB3, ErbB4, HER1, HER2, HER3, HER4, TGF, TGFB1, TGFB2, TGFB3, TGFB4, VEGF, VEGFR1, Flt-1, VEGFR2, Flk-1 / KDR, VEGFR3, Flt4, RANK, TNFRSFIIA, SLAMF, SLAMF1, SLAMF2, SLAMF3, SLAMF4, SLA MF5, SLAMF6, SLAMF7, SLAMF8, PDGFRα, PDGFRβ, KIR3DL3, KIR3DP1, KIR3DL4, KIR3DL2, MIC, MICA, MICB, TNF, LTα, LTβ, FASL, 4-IBBL, OX40L, TNF-related apoptosis-inducing ligand (TRAIL), DR1, DR2, DR3, DR4, DR5, DR6, DR7, DR8, CTLA4, PD1, CD66a, CD66b, CD66c, CD66d, CD66e , CD66f, TIM1, TIM2, TIM3, LAG, LAG3, CD2, CD3, CD19, CD20, CD22, CD25, CD27, CD30, CD33, CD38, CD39, CD52, CD70, CD73, CD94, C D134, CD137, CD252, CD340, GalNAc-O-ser / thr, Neu5Acα2-6GalNAcα-O-Ser / Thr, SSEA-, SSEA-2, SSEA-3, SSEA-4, Gal-Gal-NAc , Lewis Y, Sialyl Lewis X, Sialyl Lewis A, -Globo, -Isoglobo, -Ganglio, -Isoganglio, -Lacto, -Neolacto, LactoGanglio, -Muco, -Neogala, -Mollu, -Arthro, -Schisto, -Spirometo, GD1, GD2, GD3, GM1, GM2, Fucosyl Gm1, Neu5GcGm3, and polysialic acid.

[0042] In some embodiments, the antibody produced by the cell is selected from the group consisting of adalimumab, alemtuzumab, alirocumab, atezolizumab, avelumab, belimumab, benralizumab, bevacizumab, bezlotoxumab, blinatumomab, brentuximab vedotin, canakinumab, cantuzumab mertansine, cantuzumab vedotin, carotuximab, certolizumab pegol, cetuximab, , claudiximab, daclizumab, daratumumab, denosumab, depatuxizumab mafodotin, dinutuximab, durvalumab, elotuzumab, enfortumab vedotin, enoblitzumab, gemtuzumab ozogamicin, golimumab, guselkumab, ibalizumab, ibritumomab tiuxetan, ifavotuzumab, inebilizumab, infliximab , inotuzumab ozogamicin, ipilimumab, isatuximab, margetuximab, mepolizumab, necitumumab, nimotuzumab, nivolumab, obiltoxaximab, obinutuzumab, ocrelizumab, ofatumumab, olaratumab, omalizumab, palivizumab, pembrolizumab, pertuzumab, polatuzumab vedotin, racotumomab, ramucirumab, ranibizumab, raxibacumab , rituximab, rovalpituzumab tesirine, sacituzumab, sacituzumab govitecan, sarilumab, secukinumab, siltuximab, sirukumab, teprotumumab, tildrakizumab, tocilizumab, tositumomab, trastuzumab, trastuzumab duocarmazine, trastuzumab emtansine, ublituximab, ustekinumab, vedolizumab, or their biosimilars.

[0043] A biosimilar is an antibody that has the same or similar sequence as an antibody named herein, but may not be identical. A biosimilar antibody may also be an antibody that is so similar to an existing FDA-approved reference product, including those described herein, that there are no clinically meaningful differences. A biosimilar is clinically meaningful when there are no meaningful (significant) differences in safety, purity, and potency (safety and effectiveness) compared to the reference product.

[0044] Without wishing to be bound by any particular theory, the composition can be used to reduce the level of fucosylation of a protein in a cell by inhibiting fucosyltransferase with the composition. The fucosylation can be reduced for a protein produced by the cell. In some embodiments, the protein to be reduced in fucosylation is an antibody. Non-limiting examples of antibodies are described herein. In some embodiments, the composition reduces the level of fucosylation by about 1% to about 99%. In some embodiments, the composition reduces the level of fucosylation by about 1% to about 95%. In some embodiments, the composition reduces the level of fucosylation by about 1% to about 90%. In some embodiments, the composition reduces the level of fucosylation by about 1% to about 85%. In some embodiments, the composition reduces the level of fucosylation by about 1% to about 80%. In some embodiments, the composition reduces the level of fucosylation by about 1% to about 75%. In some embodiments, the composition reduces the level of fucosylation by about 1% to about 70%. In some embodiments, the composition reduces the level of fucosylation by about 1% to about 65%. In some embodiments, the composition reduces the level of fucosylation by about 1% to about 60%. In some embodiments, the composition reduces the level of fucosylation by about 1% to about 55%. In some embodiments, the composition reduces the level of fucosylation by about 1% to about 50%. In some embodiments, the composition reduces the level of fucosylation by about 1% to about 45%. In some embodiments, the composition reduces the level of fucosylation by about 1% to about 40%. In some embodiments, the composition reduces the level of fucosylation by about 1% to about 35%. In some embodiments, the composition reduces the level of fucosylation by about 1% to about 30%. In some embodiments, the composition reduces the level of fucosylation by about 1% to about 25%. In some embodiments, the composition reduces the level of fucosylation by about 1% to about 20%.In some embodiments, the composition reduces the level of fucosylation by about 1% to about 15%. In some embodiments, the composition reduces the level of fucosylation by about 1% to about 10%. In some embodiments, the composition reduces the level of fucosylation by about 1% to about 5%. In some embodiments, the composition reduces the level of fucosylation by about 5% to about 99%. In some embodiments, the composition reduces the level of fucosylation by about 10% to about 99%. In some embodiments, the composition reduces the level of fucosylation by about 15% to about 99%. In some embodiments, the composition reduces the level of fucosylation by about 20% to about 99%. In some embodiments, the composition reduces the level of fucosylation by about 25% to about 99%. In some embodiments, the composition reduces the level of fucosylation by about 30% to about 99%. In some embodiments, the composition reduces the level of fucosylation by about 35% to about 99%. In some embodiments, the composition reduces the level of fucosylation by about 40% to about 99%. In some embodiments, the composition reduces the level of fucosylation by about 45% to about 99%. In some embodiments, the composition reduces the level of fucosylation by about 50% to about 99%. In some embodiments, the composition reduces the level of fucosylation by about 55% to about 99%. In some embodiments, the composition reduces the level of fucosylation by about 60% to about 99%. In some embodiments, the composition reduces the level of fucosylation by about 65% to about 99%. In some embodiments, the composition reduces the level of fucosylation by about 70% to about 99%. In some embodiments, the composition reduces the level of fucosylation by about 75% to about 99%. In some embodiments, the composition reduces the level of fucosylation by about 80% to about 99%. In some embodiments, the composition reduces the level of fucosylation by about 85% to about 99%. In some embodiments, the composition reduces the level of fucosylation by about 90% to about 99%. In some embodiments, the composition reduces the level of fucosylation by about 95% to about 99%.In some embodiments, the level of fucosylation of a protein or pool of proteins is reduced by at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95%. In some embodiments, the composition reduces but does not eliminate fucosylation. The reduction in protein fucosylation is based on the level of fucosylation of a protein produced from a particular cell type, and the cells are contacted with the compositions and compounds provided herein. For example, when an antibody is produced from a CHO cell, and then the same or similar CHO cells producing the antibody are contacted with the compositions and compounds provided herein, the level of fucosylation for the antibody is reduced. The reduction is compared to an antibody produced from the CHO cell that is not contacted with the compositions and compounds provided herein.

[0045] In some embodiments, a method for inhibiting or reducing protein fucosylation in a cell is provided. In some embodiments, the method comprises contacting the cell with any of the compositions or compounds provided herein. In some embodiments, the cell is contacted with the composition in vitro or in vivo. In some embodiments, the composition inhibits fucosylation by inhibiting fucosyltransferase.

[0046] In some embodiments, the cell is any of the cell types disclosed herein, although the cell types presented herein are non-limiting examples. In some embodiments, the cell produces an antibody. In some embodiments, the antibody is a recombinant antibody. In some embodiments, the antibody is directed against the following targets: IL8, ErbB, ErbB1, ErbB2, ErbB3, ErbB4, HER1, HER2, HER3, HER4, TGF, TGFB1, TGFB2, TGFB3, TGFB4, VEGF, VEGFR1, Flt-1, VEGFR2, Flk-1 / KDR, VEGFR3, Flt4, RANK, TNFRSFIIA, SLAMF, SLAMF1, SLAMF2, SLAMF3, SLAMF4, SLA MF5, SLAMF6, SLAMF7, SLAMF8, PDGFRα, PDGFRβ, KIR3DL3, KIR3DP1, KIR3DL4, KIR3DL2, MIC, MICA, MICB, TNF, LTα, LTβ, FASL, 4-IBBL, OX40L, TNF-related apoptosis-inducing ligand (TRAIL), DR1, DR2, DR3, DR4, DR5, DR6, DR7, DR8, CTLA4, PD1, CD66a, CD66b, CD66c, CD66d, CD66e , CD66f, TIM1, TIM2, TIM3, LAG, LAG3, CD2, CD3, CD19, CD20, CD22, CD25, CD27, CD30, CD33, CD38, CD39, CD52, CD70, CD73, CD94, C D134, CD137, CD252, CD340, GalNAc-O-ser / thr, Neu5Acα2-6GalNAcα-O-Ser / Thr, SSEA-, SSEA-2, SSEA-3, SSEA-4, Gal-Gal-NAc , Lewis Y, Sialyl Lewis X, Sialyl Lewis A, -Globo, -Isoglobo, -Ganglio, -Isoganglio, -Lacto, -Neolacto, LactoGanglio, -Muco, -Neogala, -Mollu, -Arthro, -Schisto, -Spirometo, GD1, GD2, GD3, GM1, GM2, Fucosyl Gm1, Neu5GcGm3, and polysialic acid.

[0047] In some embodiments, the antibody is selected from the group consisting of adalimumab, alemtuzumab, alirocumab, atezolizumab, avelumab, belimumab, benralizumab, bevacizumab, bezlotoxumab, blinatumomab, brentuximab vedotin, canakinumab, cantuzumab mertansine, cantuzumab vedotin, carotuximab, certolizumab pegol, cetuximab, claudiximab, claudiximab, daclizumab, daratumumab, denosumab, depatuxizumab mafodotin, dinutuximab, durvalumab, elotuzumab, enfortumab vedotin, enoblitzumab, gemtuzumab ozogamicin, golimumab, guselkumab, ibalizumab, ibritumomab tiuxetan, ifavotuzumab, inebilizumab, infliximab, inotuzumab Buozogamicin, ipilimumab, isatuximab, margetuximab, mepolizumab, necitumumab, nimotuzumab, nivolumab, obiltoxaximab, obinutuzumab, ocrelizumab, ofatumumab, olaratumab, omalizumab, palivizumab, pembrolizumab, pertuzumab, polatuzumab vedotin, racotumomab, ramucirumab, ranibizumab, raxibacumab, rituximab, ximab, rovalpituzumab tesilin, sacituzumab, sacituzumab govitecan, sarilumab, secukinumab, siltuximab, sirukumab, teprotumumab, tildrakizumab, tocilizumab, tositumomab, trastuzumab, trastuzumab duocarmazine, trastuzumab emtansine, ublituximab, ustekinumab, vedolizumab, or a biosimilar thereof. The level of fucosylation inhibition or reduction may be as described herein.

[0048] In some embodiments, methods of inhibiting fucosyltransferase are provided. In some embodiments, the methods include contacting the fucosyltransferase with any of the compositions or compounds provided herein. In some embodiments, the fucosyltransferase is present in a cell. In some embodiments, the cell is any of the cell types provided herein. In some embodiments, the cell produces an antibody. In some embodiments, the antibody is a recombinant antibody. In some embodiments, the antibody is any of the antibodies disclosed herein. In some embodiments, the antibody binds to any of the targets or antigens provided herein.

[0049] In some embodiments, methods of producing antibodies are provided. In some embodiments, methods of producing antibodies with reduced fucosylation are provided. In some embodiments, the methods include contacting an antibody-producing cell with any of the compositions and compounds provided herein. The cell may be contacted with the compositions and compounds in vitro or in vivo. The antibody-producing cell is not limited to any particular cell type, and may be, for example, any of the cell types provided herein. In some embodiments, the antibody is a recombinant antibody. In some embodiments, the antibody is any of the antibodies provided herein. In some embodiments, the antibody is directed against any of the antigens disclosed herein.

[0050] In some embodiments, any of the methods provided herein include purifying an antibody with reduced fucosylation. The antibody can be purified, for example, by column chromatography, precipitation, etc. Other purification methods include, but are not limited to, size exclusion chromatography, ammonium sulfate precipitation, ion exchange chromatography, immobilized metal chelate chromatography, thiophilic adsorption, Melon™ Gel chromatography, antibody purification with Protein A, G, and L antibody binding ligands, Protein A, G, and L, etc. These purification methods are non-limiting and other methods can be used.

[0051] In some embodiments, methods of producing antibodies with altered glycosylation patterns are provided. In some embodiments, the methods include contacting a cell producing the antibody with any of the compositions or compounds provided herein. In some embodiments, the composition is contacted with the cell in vitro or in vivo. In some embodiments, the cell is of any of the cell types provided herein. In some embodiments, the antibody is a recombinant antibody. In some embodiments, the antibody is any of the antibodies disclosed herein. In some embodiments, the antibody is directed against any of the antigens disclosed herein.

[0052] The antibodies produced herein can also be used in a method of treating various diseases in a subject for whom the method of treatment has been developed, such as cancer, autoimmune disease, etc. Thus, in some embodiments, a method of treating a subject is provided. In some embodiments, the method includes contacting a cell producing a therapeutic antibody with a compound or composition described herein to produce a therapeutic antibody with reduced fucosylation. Non-limiting examples of therapeutic antibodies are described herein. The reduced fucosylation antibody can be isolated and formulated into a pharmaceutical composition or formulation suitable for administration to a subject in need thereof. In some embodiments, the subject is treated for cancer or an autoimmune disease, such as rheumatoid arthritis, Crohn's disease, ulcerative colitis, ankylosing spondylitis, Behcet's disease, fistulizing disease, hidradenitis suppurativa, juvenile idiopathic arthritis, psoriasis, psoriatic arthritis, and relapsing polychondritis.

[0053] The present embodiment will now be described with reference to the following examples. These examples are presented for illustrative purposes only, and the present embodiment should not be construed as being limited to these examples in any way, but should be construed as embracing all variations that become evident as a result of the teachings provided herein. Those skilled in the art will readily recognize a variety of non-critical parameters that can be changed or modified to produce essentially similar results. EXAMPLES

[0054] The following examples are illustrative of the compositions and methods described herein, but are not intended to be limiting thereof. Other suitable modifications and adaptations known to those skilled in the art are within the scope of the following embodiments.

[0055] Example 1: Inhibition of fucosylation by selected compounds The compounds exemplified in this example were found to inhibit FUT8 activity, thereby demonstrating that the compounds are capable of inhibiting fucosyltransferases. The compounds exemplified below were found to be effective in inhibiting FUT8 activity and would be expected to reduce fucosylation of antibodies produced from cells containing fucosyltransferases such as FUT8. Briefly, the compounds were mixed with fucosyltransferase (FUT-8) and an appropriate substrate and incubated for 1 hour. The reaction was stopped and a detection reagent mixture was added to detect the amount of fucosylation of the substrate protein. The inhibition values ​​for each compound are shown in Table 2 and are also shown in Figures 1-19.

[0056] [Table 7]

[0057] Example 2: Fucosyltransferase inhibitors reduce antibody fucosylation The compounds exemplified in this example were found to reduce the level of fucosylation of antibodies produced by cells containing fucosyltransferases such as FUT8. Briefly, epicatechin monogallate (ECG), epicatechin-3-monogallate (ECGC), and aurintricarboxylic acid (ATA) were added to cell culture medium and added to CHO cells (ATCC CRL-12445) obtained from ATCC (also described in U.S. Patent No. 6,025,158, which is incorporated herein in its entirety) producing anti-IL8 IgG monoclonal antibody (mAb). After the indicated period, the cells were harvested and the anti-IL8 mAb was purified by standard methods. The purified anti-IL8 mAb was then reduced by adding dithiothreitol and subjected to FC-MS analysis to measure the level of fucosylation. The reduction in the level of fucosylation of anti-IL8 mAb is shown in Figures 20 and 21. These results indicate that antibody fucosylation is reduced by inhibiting fucosyltransferases. Furthermore, ECG showed a dose-dependent decrease in core fucosylated species with a concomitant increase in nonfucosylated species. A 25% reduction in fucosylation was observed at the highest concentration of ECG (100 μM), while a 13% reduction was achieved at a 10-fold lower concentration. A 6% reduction in fucosylation was achieved with 10 μM ECGC. For this antibody, ATA did not change the fucosylation level at either concentration tested (data not shown). These results indicate that antibody fucosylation may be reduced by using different types of fucosyltransferase inhibitors.

[0058] The present description is not limited to the specific processes, compositions, or methodologies described, as these processes, compositions, or methodologies may vary. The terms used in the description are only intended to describe a particular version or embodiment, and are not intended to limit the scope of the embodiments described herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference to those commonly understood in the art. However, in case of conflict, the patent specification, including definitions, shall prevail.

[0059] From the foregoing, it will be understood that various embodiments of the present disclosure have been described herein for purposes of illustration, and that various modifications can be made without departing from the scope and spirit of the present disclosure. Accordingly, the various embodiments disclosed herein are not intended to be limiting.

Claims

1. A method for producing an antibody having reduced fucosylation in FUT-8, the method comprising contacting a cell producing the antibody in vitro with a composition comprising a compound selected from the group consisting of epicatechin monogallate, epigallocatechin-3-monogallate, epigallocatechin-3,5-digallate, or any combination thereof.

2. The method of claim 1 , wherein the antibody is secreted from the cell.

3. The method of claim 2, wherein the compound or composition is contacted with the fucosyltransferase prior to the secretion of the antibody.

4. The method of any of claims 1 to 3, wherein the level of fucosylation of the produced antibody is reduced compared to antibodies produced from cells not contacted with the compound or composition.

5. The compound or composition is selected from the group consisting of hypotonic buffer, methanol, ethanol, acetone, toluene, DMSO, alkyltrimethylammonium bromide, saponin, Triton X-100, Tween-20, Tween-80, digitonin, sodium dodecyl sulfate (SDS), beta-hemolytic cytolysin, streptolysin-O (SLO), perfringolysin-O (PFO), or any combination thereof. The method of any one of claims 1 to 4, further comprising at least one cell permeabilizing agent.

6. The method of any one of claims 1 to 5, wherein the cell is a CHO cell, an NSO cell, an Sp2 / 0 cell, an HEK293 cell, or a PER.C6 cell.

7. The method of any one of claims 1 to 6, wherein the antibody binds to one or more of the following targets: IL8, ErbB, ErbB1, ErbB2, ErbB3, ErbB4, HER1, HER2, HER3, HER4, TGF, TGFB1, TGFB2, TGFB3, TGFB4, VEGF, VEGFR1, Flt-1, VE GFR2, Flk-1 / KDR, VEGFR3, Flt4, RANK, TNFRSFIIA, SLAMF, SLAMF1, SLAMF2, SLAMF3, SLAMF4, SLAMF5, SLAMF6, SLAMF7, SLAMF 8, PDGFRα, PDGFRβ, KIR3DL3, KIR3DP1, KIR3DL4, KIR3DL2, MIC, MICA, MICB, TNF, LTα, LTβ, FASL, 4-IBBL, OX40L, TNF-related apoptosis-inducing ligand (TRAIL), DR1, DR2, DR3, DR4, DR5, DR6, DR7, DR8, CTLA4, PD1, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, TIM1, TIM 2, TIM3, LAG, LAG3, CD2, CD3, CD19, CD20, CD22, CD25, CD27, CD30, CD33, CD38, CD39, CD52, CD70, CD73, CD94, CD134, CD137, CD252, CD340, GalNAc-O-ser / thr, Neu5Acα2-6GalNAcα-O-ser / thr, SSEA-, SSEA-2, SSEA-3, SSEA-4, Gal-Gal-NAc, Lewis Y, Si The method of the present invention further comprises administering to the patient an amino acid sequence selected from the group consisting of allyl Lewis X, sialyl Lewis A, -Globo, -Isoglobo, -Ganglio, -Isoganglio, -Lacto, -Neolacto, LactoGanglio, -Muco, -Neogala, -Mollu, -Arthro, -Schisto, -Spirometo, GD1, GD2, GD3, GM1, GM2, Fucosyl Gm1, Neu5GcGm3, and polysialic acid.

8. The method according to any one of claims 1 to 6, wherein the antibody produced by the cells is selected from the group consisting of adalimumab, alemtuzumab, alirocumab, atezolizumab, avelumab, belimumab, benralizumab, bevacizumab, bezlotoxumab, blinatumomab, brentuximab vedotin, burosumab, canakinumab, cantuzumab mertansine, cantuzumab ravtansine, carotuximab, certolizumab pegol, cetuximab, claudiximab, daclizumab, daratumumab, denosumab, depatuxizumab mafodotin, dinutuximab, durvalumab, elotuzumab, enfortumab vedotin, enoblitzumab, and gemtuzumab. Ozogamicin, golimumab, guselkumab, ibalizumab, ibritumomab tiuxetan, ifavotuzumab, inebilizumab, infliximab, inotuzumab ozogamicin, ipilimumab, isatuximab, margetuximab, mepolizumab, necitumumab, nimotuzumab, nivolumab, obiloxaximab, obinutuzumab, ocrelizumab, ofatumumab, olaratumab, omalizumab, palivizumab, pembrolizumab, pertuzumab, polatuzumab vedotin, racotumomab, ramucirumab, ranibizumab, raxibacumab, rituximab, rovalpituzumab The therapeutic agent is selected from the group consisting of tecilin, sacituzumab, sacituzumab govitecan, sarilumab, secukinumab, siltuximab, sirukumab, teprotumumab, tildrakizumab, tocilizumab, tositumomab, trastuzumab, trastuzumab duocarmazine, trastuzumab emtansine, ublituximab, ustekinumab, vedolizumab, or a biosimilar thereof.

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