Use of Fucosylation Inhibitors to Generate Defucosylated Antibodies

Abstract

The present invention provides an inhibitor of fucosylation during protein expression from mammalian cells. The inhibitor is derived from rhamnose and acts by inhibiting GDP-mannose 4,6-dehydratase (GMD). The present invention further provides methods for producing proteins, e.g., antibodies, with reduced levels of fucosylation, and antibodies produced by the methods of the present invention. Such hypofucosylated or nonfucosylated antibodies can be used in the treatment of human diseases where it would be therapeutically beneficial to use hypofucosylated or nonfucosylated anti-CTLA-4 antibodies to direct antibody-dependent cellular cytotoxicity (ADCC)-mediated killing of cells expressing the antibody's target on their surface, for example, upon depletion of Tregs in cancer patients.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 951,318, filed December 20, 2019, the disclosure of which is incorporated herein by reference.

[0002] Sequence Listing The sequence listing submitted electronically herewith is also incorporated herein by reference in its entirety (Filename: 202001104_SEQL_13347WOPCT_GB.txt; Creation Date: November 4, 2020; File Size: 11 KB). [Background technology]

[0003] 2. Background of the Invention Therapeutic antibodies are increasingly commonly used to treat human diseases. Antibodies that bind therapeutic targets are created, selected, and modified to have the desired effect on disease mechanisms. The treatment of autoimmune diseases has been revolutionized by the use of antibodies that bind to inflammatory mediators such as cytokines and their receptors. Such antibodies typically aim simply to block inflammatory signaling pathways and only need to bind to a target protein with an epitope that blocks binding to its ligand or receptor.

[0004] Antibodies are also being developed for the treatment of cancer. The original therapeutic model for anti-cancer antibodies was the idea of ​​a "magic bullet" that would specifically target toxic drugs to tumor cells. Antibodies are generated against tumor-specific cell surface antigens and then derivatized with a cytotoxic "payload" (often a traditional chemotherapy drug). When administered to a cancer patient, the antibody circulates, binds specifically to tumor cells, and delivers the toxic payload only to the tumor cells, sparing healthy tissues significantly, thereby reducing side effects. Drugs can be attached by linkers that release cytotoxins near the target tumor cells to create locally high concentrations within the tumor, or they can remain attached to the antibody until internalization after the antibody binds to the cell surface.

[0005] An alternative to cytotoxic payloads is the use of antibodies that can direct a mounted immune response specifically against tumor cells. Similar to the magic bullet approach, antibodies can direct cytotoxicity against tumor cells, but in this case, they direct a cytotoxic immune response. Such antibodies not only bind to tumor-specific cell surface markers, but also target anti-tumor CD8 + They need to be designed to attract and / or activate immune cells, such as T cells.

[0006] A very recent approach to the treatment of cancer with antibodies is immuno-oncology. In this approach, antibodies are designed not to kill tumor cells directly but to modify the activity of the immune system to generate an effective anti-tumor immune response. Many tumors induce an anti-tumor immune response, but this immune response has been found to be impeded by the activity of various cell surface receptors that block signals that activate the anti-tumor response or enhance immune suppressive mechanisms. Although immune suppressive mechanisms are essential to restore homeostasis and otherwise limit immune responses after they are no longer needed, these mechanisms can suppress anti-tumor immune responses when such responses would be beneficial. One such immune suppressor is regulatory T cells (Tregs), a subset of T cells that function to suppress the activity of cytotoxic CD8+ T cells. In patients with life-threatening tumors, such suppressive effects can allow tumors to grow that might otherwise be eliminated or controlled. Indeed, the presence of high levels of Tregs within a tumor is a known marker for poor prognosis. Tao et al. (2012) Lung Cancer 75:95.

[0007] As a result, in the treatment of some cancers, it is beneficial to deplete the Treg population to allow an unfettered antitumor immune response. As with tumor cells, one approach is to use antibodies specific for Tregs, such as anti-CTLA-4 or anti-CCR4. Such antibodies are designed to deplete Tregs and may do so by inducing an immune response against these cells, for example, by antibody-dependent cellular cytotoxicity (ADCC) mediated by CD8+ T cells. Antibodies are designed with an Fc region that binds to activating Fc receptors on T cells to enhance the antitumor immune response. Such antibodies are said to have effector functions. Effector functions can be enhanced by modifying the Fc portion of the antibody that interacts with immune cells, such as by modifying the amino acid sequence of the Fc region or by modifying glycosylation.

[0008] It has also been found that removal of fucose from N-linked glycan chains at N297 of human immunoglobulin heavy chains enhances binding to activating Fc receptors, thereby greatly enhancing antitumor ADCC-mediated cytotoxicity. Rothman et al. (1989) Mol. Immunol. 26:1113, at 1122 (proposing reduction of antibody core fucosylation to enhance ADCC of antibodies used in tumor immunotherapy); Harris et al. (1997) Biochemistry 36:1581; Satoh et al. (2003) Expert Opin. Bio. Ther. 6:1161.

[0009] Several methods are known for producing antibodies with reduced fucosylation, including hypofucosylated and non-fucosylated antibodies (Le et al. (2016) Biochim. Biophys. Acta 1860:1655.). Antibodies can be produced in cell lines that are naturally devoid of fucosylation (Lifely et al. (1995) Glycobiology 5:813) or in cell lines in which key enzymatic components of the fucosylation pathway have been knocked out, e.g., cells lacking fucosyltransferase 8 (FUT8), such as POTELLIGENT® Chinese hamster ovary (CHO) cells. See, e.g., Rothman et al. (1989) Mol. Immunol. 26:1113; WO97 / 27303; WO99 / 54342; WO00 / 61739; WO02 / 31140. Alternatively, inhibitors of the enzymatic fucosylation pathway can be added to the culture during antibody production.See, for example, Rothman et al. (1989) Mol. Immunol. 26:1113; U.S. Patent No. 8,071,336; WO09 / 135181; WO14 / 130613; EP2958905B1; Allen et al. (2016) ACS Chem. Biol. 11:2734.Exemplary small molecule inhibitors of fucosylation include, but are not limited to, castanospermine, 2F-peracetyl-fucose, 2-deoxy-2-fluoro-L-fucose, 6,6,6-trifluorofucose (fucostatin I) and 6,6,6-trifluorofucose phosphonate analog (fucostatin II).Rothman et al. (1989) Mol. Immunol. 26:1113; Okeley et al. (2016) Proc. Nat'l Acad. Sci. (USA) 110:5404; Rillahan et al. (2012) Nat. Chem. Biol. 8:661; U.S. Patent No. 8,163,551; EP2958905B1; Allen et al. (2016) ACS Chem. Biol. 11:2734. Other creative approaches include GLYMAXX® fucosylation inhibition technology, which is the enzymatic depletion of GDP-fucose precursors in antibody-producing cell lines. See, e.g., U.S. Patent No. 8,642,292; von Horsten et al. (2010) Glycobiology 20:1607; Roy et al. (2018) mAbs 10:416.

[0010] There is a need for hypofucosylated and nonfucosylated antibodies, as well as improved methods for producing them. A method that can adjustably increase or decrease the proportion of molecules with fucosylation is particularly valuable in exploratory research. Ideally, such a method would not require the introduction of gene constructs into the cell line used for antibody production, or the time-consuming creation of new stable cell lines, and would not significantly reduce the titer of the antibody produced compared to the production of fucosylated antibodies. Summary of the Invention

[0011] The present invention provides compounds for use as fucosylation inhibitors that inhibit mammalian GDP-mannose 4,6-dehydratase (GMD), e.g., hamster GMD. Such compounds are used, for example, in the production of proteins, such as antibodies, that have reduced fucosylation of N-linked glycans, where the compounds are added to cell cultures during production of the protein (e.g., antibody).

[0012] In various embodiments, the compound of the invention is a derivative of rhamnose, such as GDP-D-rhamnose, Ac-GDP-D-rhamnose, or sodium rhamnose phosphate. In one embodiment, GDP-D-rhamnose is a compound of the invention. In another embodiment, Ac-GDP-D-rhamnose is a compound of the invention. In yet another embodiment, sodium rhamnose phosphate is a compound of the invention. In various embodiments, the fucosylation inhibitor of the invention is present in the culture medium at a concentration of 6 mM or more, or at a concentration of 10 mM or more.

[0013] In another aspect, the invention provides a method for producing a protein, such as an antibody, with reduced fucosylation by including a compound of the invention in the culture medium used during production of the protein from a cell line expressing the protein (e.g., an antibody). In certain embodiments, the compound is present in the culture medium for all or substantially all of the time that the protein (e.g., an antibody) to be isolated is produced by the cell line to maximize the proportion of non-fucosylated protein (e.g., an antibody) produced, although as a general rule, the compound need only be present in the production culture sufficient to achieve the desired level of non-fucosylation.

[0014] In a further aspect, the invention provides proteins with reduced fucosylation produced by the methods of the invention, such as proteins with reduced fucosylation (e.g., ≧20% or ≧40% defucosylated polypeptide chains), or hypofucosylated or nonfucosylated proteins.

[0015] In related aspects, the invention provides antibodies with reduced fucosylation produced by the methods of the invention, e.g., antibodies that exhibit a 2-fold or greater enhancement of ADCC compared to the same antibody produced in the same cell line in the absence of a fucosylation inhibitor (as determined by the methods described in Example 2), and / or proteins with reduced fucosylation (e.g., ≧20% or ≧40% defucosylated polypeptide chains), or hypofucosylated or nonfucosylated proteins.

[0016] In yet a further aspect, the invention provides a method of treating a human disease (e.g., cancer) by administering to a patient in need thereof an antibody or other protein with reduced fucosylation produced by the methods of the invention.

[0017] In various embodiments, the compounds of the invention are included in cell growth medium used during antibody production at a concentration of 1 mM, 2 mM, 3 mM, 6 mM, 10 mM or more.

[0018] Exemplary antibodies that can be made in a hypofucosylated or nonfucosylated form by the methods of the invention include antibodies that bind to human CD20, CCR4, EGFR, CD19, Her2, IL-5R, CD40, BCMA, Siglec 8, CD147, CD30, EphA3, Fucosyl GM1, CTLA-4, MICA, and ICOS. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 shows the structures of three compounds, specifically, GDP-D-rhamnose (Formula I), Ac-GDP-D-rhamnose (Formula II), and sodium rhamnose phosphate (Formula III). [Figure 2A] Figure 2A shows the percentage of nonfucosylated antibody relative to antibody grown in the presence or absence of an exemplary fucosylation inhibitor of the invention at concentrations between 1 mM and 6 mM. The structures of GDP-D-rhamnose and Ac-GDP-D-rhamnose are shown in Figure 1. [Figure 2B] Figure 2B shows the antibody titers of the same antibody preparations as in Figure 2A. Ac-GDP-D-rhamnose is as effective as GDP-D-rhamnose in increasing the proportion of nonfucosylated antibodies with less adverse effect on titer (yield). [Figure 3A]3A, 3B, and 3C show electropherograms of antibody preparations made with cells cultured without fucosylation inhibitors or in the presence of 6 mM or 10 mM Ac-GDP-D-rhamnose, respectively. Peaks of different glycoforms are shown, with white boxes indicating fucosylated species and dark boxes indicating non-fucosylated species. With increasing concentrations of Ac-GDP-D-rhamnose, the proportion of non-fucosylated species increases. [Figure 3B] 3A, 3B, and 3C show electropherograms of antibody preparations made with cells cultured without fucosylation inhibitors or in the presence of 6 mM or 10 mM Ac-GDP-D-rhamnose, respectively. Peaks of different glycoforms are shown, with white boxes indicating fucosylated species and dark boxes indicating non-fucosylated species. With increasing concentrations of Ac-GDP-D-rhamnose, the proportion of non-fucosylated species increases. [Figure 3C] 3A, 3B, and 3C show electropherograms of antibody preparations made with cells cultured without fucosylation inhibitors or in the presence of 6 mM or 10 mM Ac-GDP-D-rhamnose, respectively. Peaks of different glycoforms are shown, with white boxes indicating fucosylated species and dark boxes indicating non-fucosylated species. With increasing concentrations of Ac-GDP-D-rhamnose, the proportion of non-fucosylated species increases. [Figure 4A] 4A and 4B provide exemplary synthetic schemes for producing rhamnose phosphate, GDP-d-rhamnose and Ac-GDP-d-rhamnose. See Example 1. [Figure 4B] 4A and 4B provide exemplary synthetic schemes for producing rhamnose phosphate, GDP-d-rhamnose and Ac-GDP-d-rhamnose. See Example 1. [Figure 5A] 5A, 5B and 5C provide a second exemplary synthetic scheme for producing GDP-d-rhamnose and Ac-GDP-d-rhamnose. [Figure 5B] 5A, 5B and 5C provide a second exemplary synthetic scheme for producing GDP-d-rhamnose and Ac-GDP-d-rhamnose. [Figure 5C] 5A, 5B and 5C provide a second exemplary synthetic scheme for producing GDP-d-rhamnose and Ac-GDP-d-rhamnose. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] Detailed Description of the Invention definition In order that this disclosure may be more readily understood, certain terms are first defined. As used in this application, unless expressly defined otherwise herein, each of the following terms shall have the meaning set forth below. Further definitions are set forth in this application.

[0021] "Administering" refers to the physical introduction of a composition comprising a therapeutic agent into a subject using any of a variety of methods and delivery systems known to those skilled in the art. Preferred routes of administration for the antibodies of the invention include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, for example, by injection or infusion. The term "parenteral administration" as used herein generally refers to modes of administration other than enteral and topical administration by injection, and includes intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intraarticular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intradural and intrasternal injection and infusion, as well as in vivo electroporation. Alternatively, the antibodies of the invention can be administered via a parenteral route, for example, by topical, epidermal or mucosal routes of administration, such as intranasal, oral, vaginal, rectal, sublingual or topical. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods of time. Administration may be performed by one or more individuals, including, but not limited to, a doctor, a nurse, another health care provider, or the patient themselves. As claimed, a "patient in need thereof" refers to any human subject diagnosed with the disease being treated, such as, for example, cancer.

[0022] "Antibody" (Ab) includes, but is not limited to, immunoglobulin glycoproteins that specifically bind to an antigen and contain at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Antibodies produced by the methods of the invention, including production of antibodies in cell lines cultured in the presence of fucosylation inhibitors of the invention, are referred to as antibodies of the invention. In conventional antibodies, each H chain contains a heavy chain variable region (herein referred to as V H The heavy chain constant region is abbreviated as C H1 , C H2 , and C H3 Each light chain comprises three domains: a light chain variable region (referred to herein as V L The light chain constant region comprises one domain, C L It is composed of: V H and VL The region can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). H and V L is composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens.

[0023] As used herein and in accordance with conventional interpretation, an antibody described as comprising "a" heavy chain and / or "a" light chain means an antibody comprising "at least one" of the described heavy and / or light chains, and thus encompasses antibodies having two or more heavy and / or light chains. Specifically, an antibody thus described encompasses conventional antibodies having two substantially identical heavy chains and two substantially identical light chains. The antibody chains may be substantially identical, but not completely identical, if they differ due to post-translational modifications such as C-terminal truncation of lysine residues, alternative glycosylation patterns, etc. An "antibody" may also comprise two different antigen-binding domains, e.g., a bispecific antibody or an antibody that binds to two different epitopes on the same target, and thus may comprise two non-identical heavy and / or light chains.

[0024] Unless otherwise indicated or clear from the context, an antibody defined by its target specificity (e.g., an "anti-CTLA-4 antibody") means an antibody that can bind to its human target (e.g., human CTLA-4). Such an antibody may or may not bind to CTLA-4 from other species.

[0025] Immunoglobulins may be derived from any of the commonly known isotypes, including, but not limited to, IgA, secretory IgA, IgG, and IgM. IgG isotypes may be divided into subclasses in certain species: IgG1, IgG2, IgG3, and IgG4 in humans, and IgG1, IgG2a, IgG2b, and IgG3 in mice. IgG antibodies may be referred to herein by the symbol gamma (γ) or simply "G", as the context dictates; for example, IgG1 may be represented as "γ1" or "G1". "Isotype" refers to the antibody class (e.g., IgM or IgG1) encoded by the heavy chain constant region genes. "Antibody" includes, by way of example, both naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; fully synthetic antibodies; and single-chain antibodies. Unless otherwise indicated or clear from the context, the antibodies disclosed herein are human IgG1 antibodies.

[0026] An "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigen specificities (e.g., an isolated antibody that specifically binds to CTLA-4 is substantially free of antibodies that specifically bind to antigens other than CTLA-4). However, an isolated antibody that specifically binds to CTLA-4 may cross-react with other antigens, such as CTLA-4 molecules from different species. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals. In comparison, "isolated" nucleic acids refer to nucleic acid compositions of matter that are significantly different, i.e., have different chemical identities, properties and usefulness, from the nucleic acids in which they occur in nature. For example, isolated DNA, unlike naturally occurring DNA, is an independent piece of naturally occurring DNA, and not a part that is included with the larger structural complexes found in nature, chromosomes. Furthermore, isolated DNA, unlike naturally occurring DNA, can be used as a PCR primer or hybridization probe to detect biomarker genes or mutations to measure gene expression, diagnose disease, or predict the effectiveness of treatment, among other things. An isolated nucleic acid can also be purified so that it is substantially free of other cellular components or other contaminants, such as other cellular nucleic acids or proteins, using standard techniques well known in the art.

[0027] The term "monoclonal antibody" ("mAb") refers to a preparation of antibody molecules of single molecular composition, i.e., which are essentially identical in primary sequence and which display a single binding specificity and affinity for a particular epitope. Monoclonal antibodies may be produced by hybridoma, recombinant, transgenic, or other techniques known in the art.

[0028] A "human" antibody (HuMAb) refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse, etc.) have been grafted onto human framework sequences. The terms "human" antibody and "fully human" antibody are used synonymously.

[0029] "Antibody fragment" generally refers to a portion of a whole antibody, including an "antigen-binding portion" of an intact antibody ("antigen-binding fragment") that retains the ability to specifically bind to the antigen bound by the intact antibody or the Fc region of an antibody that retains FcR binding ability. Exemplary antibody fragments include Fab fragments and single-chain variable domain (scFv) fragments.

[0030] "Antibody-dependent cell-mediated cytotoxicity" ("ADCC") refers to an in vitro or in vivo cell-mediated reaction in which nonspecific cytotoxic cells expressing FcR (e.g., natural killer (NK) cells, macrophages, eosinophils and eosinophils) recognize antibodies bound to surface antigens on target cells, and subsequently cause lysis of the target cells. In principle, any effector cell that activates FcR can be the trigger to mediate ADCC.

[0031] "Cancer" refers to a broad group of diverse diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and proliferation can result in the formation of malignant tumors or cells that invade adjacent tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream.

[0032] "Cell surface receptor" means molecules and complexes of molecules that are capable of receiving a signal and transmitting such a signal across a cell membrane.

[0033] "Effector cell" refers to a cell of the immune system that expresses one or more FcRs and mediates one or more effector functions. Preferably, the cell expresses at least one type of activating Fc receptor, e.g., human FcγRIII, and is responsible for ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), NK cells, monocytes, macrophages, neutrophils, and eosinophils.

[0034] "Effector function" refers to the interaction of the Fc region of an antibody with an Fc receptor or ligand, or the biochemical event resulting therefrom. Exemplary "effector functions" include Clq binding, complement-dependent cytotoxicity (CDC), Fc receptor binding, FcγR-mediated effector functions, such as ADCC and antibody-dependent cell-mediated phagocytosis (ADCP), and down-regulation of cell surface receptors (e.g., B cell receptor; BCR). Such effector functions generally require the Fc region to bind to a binding domain (e.g., an antibody variable domain).

[0035] An "Fc receptor" or "FcR" is a receptor that binds to the Fc region of an immunoglobulin. FcRs that bind IgG antibodies include the FcγR family of receptors, including allelic variants and alternatively spliced ​​forms of these receptors. The FcγR family is composed of three activating (FcγRI, FcγRIII, and FcγRIV in mice, and FcγRIA, FcγRIIA, and FcγRIIIA in humans) receptors and one inhibitory (FcγRIIB) receptor. The various properties of human FcγRs are summarized in Table 1. Many inactive effector cell types co-express one or more activating FcγRs and inhibitory FcγRIIB, while natural killer (NK) cells selectively express one activating Fc receptor (FcγRIII in mice, FcγRIIIA in humans) but do not express inhibitory FcγRIIB in mice or humans.

[0036] "Fc region" (fragment crystallizable region), "Fc domain," or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors located on various cells of the immune system (e.g., effector cells) or binding to the first component (C1q) of the classical complement system. Thus, an Fc region is a polypeptide comprising the constant region of an antibody excluding the first constant region immunoglobulin domain. In IgG, IgA, and IgD antibody isotypes, the Fc region comprises the second (C1q) of the two heavy chains of the antibody. H2 ) and the third (C H2 The Fc regions of IgM and IgE are composed of two identical protein fragments derived from the constant domains of the heavy chain (C H In IgG, the Fc region comprises immunoglobulin domains Cγ2 and Cγ3, as well as the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is usually defined to stretch from an amino acid residue at position C226, or from an amino acid residue at position P230, to the carboxy-terminus of the heavy chain (numbering is according to the EU index, as in Kabat). The C of the Fc region of human IgG H2The domain extends from about amino acid 231 to about amino acid 340, but contains H3 The domain is C in the Fc region. H2 The Fc region is located at the C-terminal end of the domain, i.e., it extends from about amino acid 341 to about amino acid 447 of IgG. As used herein, the Fc region may be a native sequence Fc or a variant Fc. Fc can also refer to this region in isolation or in the context of a protein polypeptide that contains Fc, such as an "Fc region-containing binding protein," also referred to as an "Fc fusion protein" (e.g., an antibody or immunoadhesin). Table 1 Characteristics of human FcγR [Table 1]

[0037] "Fucosylation" as used herein means the presence of a fucose residue bisecting the innermost GlcNac residue of an N-linked glycan chain on a protein, unless otherwise specified. Although fucosylation is a bulk property of a population of protein molecules, the term may be used in reference to individual proteins within the population. For example, an individual antibody may be "fucosylated" on both heavy chains (fucosylated), neither heavy chain (non-fucosylated), or only one of the two heavy chains (hemifucosylated). A population of antibodies, e.g., a preparation from a production process, may contain a mixture of each fucosylated, non-fucosylated, and hemifucosylated antibodies and therefore may exhibit any degree of fucosylation from 0% to 100%. Percentage of fucosylation, as used herein, means the percentage of all possible fucosylation sites at which fucose is present. For example, a preparation of pure hemifucosylated antibodies is 50% fucosylated. An exemplary method for determining the percent fucosylation in an antibody preparation is described in Example 2.

[0038] GMD means "GDP-mannose 4,6-dehydratase" derived from a mammal, such as a hamster or human. GMD is referred to as Enzyme Commission (EC) number 4.2.1.47. Human GMD is also referred to as GMDS and SDR3E1. GMD catalyzes the conversion of GDP-mannose to GDP-4-keto-6-deoxymannose, the first step in the synthesis of GDP-fucose from GDP-mannose, using NADP+ as a cofactor. Unless otherwise stated or clear from the context, the term GMD in this specification refers to hamster GMD, but in most contexts both hamster and human proteins are included. Hamster (Cricetulus griseus) GMD is further described in Gene ID number: 100689436. The sequence of hamster GMD (NP_001233625.1) including the 23 amino acid signal sequence is provided in SEQ ID NO: 1 and the coding DNA sequence NM_001246696.1 is provided in SEQ ID NO: 2. Human (Homo sapiens) GMD is further described in Gene ID No: 2762 and MIM (Mendelian Inheritance in Man): 602884. The sequence of human GMD isoform 1 (NP_001491.1) including the 23 amino acid signal sequence is provided in SEQ ID NO: 3 and the coding DNA sequence NM_001500.4 is provided in SEQ ID NO: 4. Hamster and human GMD polypeptides share 98% sequence similarity and >99% sequence identity for the 347 aa mature protein.

[0039] "Immune response" refers to a biological response in a vertebrate to foreign substances, which response protects the organism against these substances and the diseases caused by them. The immune response is mediated by the action of cells of the immune system (such as, for example, T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, or neutrophils) and soluble macromolecules (including antibodies, cytokines, complement) produced either by these cells or by the liver, which selectively target, bind, damage, destroy, and / or eliminate invading pathogens, pathogen-infected cells or tissues, cancer cells or other abnormal cells, or normal human cells or tissues in the case of autoimmune disease or pathological inflammation, within the vertebrate body.

[0040] "Immune modulator" or "immunoregulator" refers to a component of a signaling pathway that may be involved in the modification, regulation, or alteration of an immune response. "Modifying," "regulating," or "altering" an immune response refers to any change in the cells of the immune system or the activity of such cells. Such modulation includes stimulation or suppression of the immune system, which may be manifested by an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, or other changes that may occur within the immune system. Both inhibitory and stimulatory immune modulators have been identified, some of which may function enhanced in the tumor microenvironment. In a preferred embodiment of the disclosed invention, the immune modulator is localized on the surface of a T cell. An "immunomodulatory target" or "immunoregulatory target" is an immune modulator that is targeted for binding by a substance, agent, moiety, compound, or molecule, and whose activity is altered by such binding. Immune modulator targets include, for example, receptors on the cell surface ("immunoregulatory receptors") and receptor ligands ("immunoregulatory ligands").

[0041] "Immunotherapy" means the treatment of a subject suffering from a disease or at risk of suffering from or recurring with a disease by methods involving inducing, enhancing, suppressing or modulating the immune response.

[0042] "Enhancing an endogenous immune response" means increasing the effectiveness or potency of an existing immune response in a subject. This increase in effectiveness and potency can be achieved, for example, by addressing mechanisms that suppress the endogenous host immune response or by stimulating mechanisms that enhance the endogenous host immune response.

[0043] "Protein" means a chain (without an upper limit on the length of the chain) that includes at least two consecutively linked amino acid residues. One or more amino acid residues in a protein may include modifications, such as, but not limited to, glycosylation, phosphorylation, or formation of disulfide bonds. The term "protein" is used interchangeably with "polypeptide" herein. A "protein" may include two or more polypeptide chains, which may include different polypeptide sequences, such as heavy and light chains of an antibody. A conventional full-length antibody includes two heavy chains and two light chains and is a "protein." A cell or cell line that expresses a "protein" that includes two or more polypeptides with different sequences expresses all chains of the protein, e.g., both heavy and light chains of an antibody.

[0044] "Protein," as used herein, includes N-linked glycans, unless otherwise indicated, with respect to the compounds and methods of the invention for producing proteins with reduced fucosylation. Proteins with N-linked glycosylation, such as Fc region (N297) glycosylation of antibodies, may be used with the compounds and methods of the invention to limit or inhibit the addition of fucose residues, which are typically added to the innermost GlcNac residue of the glycan.

[0045] As is conventional, the term "protein" (such as, for example, an "antibody") can refer to either a population of protein molecules in a preparation or each protein molecule within that population, depending on the context. For clarity, the term "defucosylated" is used herein to refer to each protein (e.g., an antibody chain) that lacks N-linked fucose, and "nonfucosylated" is used herein to refer to a population or preparation of protein molecules. As a result, each polypeptide chain may be fucosylated or defucosylated, but a population of proteins may be nonfucosylated to a certain percentage of defucosylation. Thus, a reference to a protein in terms of fucosylation levels, such as an "antibody with reduced fucosylation," necessarily refers to a heterogeneous population of protein molecules, even if not explicitly stated.

[0046] Unless otherwise stated or clear from the context, the numbering of amino acid residues in an antibody Fc region follows EU numbering conventions (EU index as in Kabat et al. (1991) Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, MD; see also Figures 3c-3f of US Patent Application Publication No. 2008 / 0248028) unless specifically referring to residues in a sequence in the sequence listing, in which case the numbering is necessarily consecutive. For example, references to the effect of amino acid substitutions in an Fc region typically use EU numbering, which allows a given residue in an antibody Fc region to be referred to by the same number regardless of the length of the variable domain to which it is attached. In rare cases, it may be necessary to consult the referencing document to ascertain the exact Fc residue being referenced.

[0047] "Rhamnose" means D-rhamnose, unless otherwise specified.

[0048] A "subject" includes a human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrates, such as non-human primates, sheep, dogs, rabbits, rodents, such as mice, rats, and guinea pigs, birds, such as chickens, amphibians, and reptiles. In a preferred embodiment, the subject is a mammal, such as a non-human primate, sheep, dog, cat, rabbit, ferret, or rodent. In a more preferred embodiment of the disclosed aspects of the invention, the subject is a human. The terms "subject" and "patient" are used interchangeably herein.

[0049] "Treatment" or "therapy" of a subject means any type of intervention or method performed on a subject, or the administration of an active agent to a subject, to reverse, alleviate, relieve, inhibit, delay, or prevent the onset, progression, development, severity, or recurrence of a symptom, complication, condition, or biochemical manifestation associated with a disease.

[0050] Conventional methods for reducing antibody fucosylation The interaction of antibodies with FcγR can be enhanced by modifying the glycan moiety attached to each Fc fragment at the N297 residue. In particular, the absence of core fucose residues strongly enhances ADCC by improving IgG binding to activating FcγRIIIA without altering antigen binding or CDC (Natsume et al. (2009) Drug Des. Devel. Ther. 3:7). There is compelling evidence that defucosylated tumor-specific antibodies lead to enhanced therapeutic activity in mouse models in vivo (Nimmerjahn & Ravetch (2005) Science 310:1510; Mossner et al. (2010) Blood 115:4393).

[0051] Modification of the glycosylation of antibodies has previously been performed, for example, by expressing the antibodies in host cells with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art. For example, cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene FUT8 (α-(1,6) fucosyltransferase (see U.S. Patent Publication No. 20040110704; Yamane-Ohnuki et al. (2004) Biotechnol. Bioeng. 87: 614), such that antibodies expressed in these cell lines lack fucose on their sugars. EP1176195 also describes cell lines with a functionally disrupted FUT8 gene, as well as cell lines that have little or no activity to add fucose to N-acetylglucosamine attached to the Fc region of antibodies, such as the rat myeloma cell line YB2 / 0 (ATCC 61363). PCT Publication No. WO03 / 035835 describes a mutant CHO cell line, Lec13, that has a reduced ability to attach fucose to Asn(297)-linked carbohydrate, resulting in hypofucosylation of antibodies expressed in the host cells. Shields et al. (2002) J. Biol. Chem. 277:26733. As described in PCT Publication No. WO2006 / 089231, antibodies with modified glycosylation profiles can also be produced in chicken eggs. Alternatively, antibodies with modified glycosylation profiles can be produced in plant cells, such as Lemna. See, for example, US Publication No. 2012 / 0276086. PCT Publication No. WO99 / 54342 describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(1,4)-N-acetylglucosamine transferase III (GnTIII)), and antibodies expressed in the engineered cell lines exhibit increased biantennary GlcNac structures that result in increased ADCC activity of the antibodies. See also Umana et al. (1999) Nat. Biotech. 17:176. Alternatively, the fucose residues of the antibodies can be cleaved off using a fucosidase enzyme.For example, the enzyme α-L-fucosidase removes fucosyl residues from antibodies (Tarentino et al. (1975) Biochem. 14:5516). Antibodies with reduced fucosylation can also be produced in cells carrying a recombinant gene encoding an enzyme that uses GDP-6-deoxy-D-lyxo-4-hexylose as a substrate, e.g., GDP-6-deoxy-D-lyxo-4-hexylose reductase (RMD), as described in U.S. Pat. No. 8,642,292. Alternatively, cells can be grown in medium containing a fucose analog that inhibits the addition of fucose residues to N-linked glycans or glycoproteins (e.g., antibodies) produced by cells grown in the medium (U.S. Pat. No. 8,163,551; WO09 / 135181). Such compounds include, but are not limited to, peracetylfucose, 6,6,6-trifluorofucose per-O-acetate, 6,6,6-trifluorofucose (fucostatin I), and a fucose-1-phosphate analog (fucostatin II).

[0052] Rhamnose derivatives as fucosylation inhibitors. In one embodiment, the present invention provides rhamnose-derived compounds, such as GDP-D-rhamnose and its derivatives, that inhibit the fucosylation of proteins produced in mammalian cell culture. Without intending to be limited by theory, such compounds may act as inhibitors of GDP-mannose-4,6-dehydratase (GMD). Exemplary compounds of the present invention include GDP-D-rhamnose (Formula I), Ac-GDP-D-rhamnose (Formula II), and sodium rhamnose phosphate (Formula III), the structures of which are set forth in FIG. 1. Exemplary methods of synthesis of the compounds of the present invention are set forth in FIGS. 4A and 4B (for Ac-GDP-D-rhamnose) and in FIGS. 4A, 4B, and 4C (for GDP-D-rhamnose), and are described in more detail in Example 1. A second exemplary method of synthesis of the compounds of the present invention is set forth in FIGS. 5A and 5B (for Ac-GDP-D-rhamnose) and in FIGS. 5A, 5B, and 5C (for GDP-D-rhamnose).

[0053] The invention also provides methods for producing proteins with reduced fucosylation, and hypofucosylated and nonfucosylated proteins, such as antibodies, by growing protein-producing cells in medium containing a fucosylation inhibitor of the invention, such as GDP-D-rhamnose, Ac-GDP-D-rhamnose, and sodium rhamnose phosphate, for example, at a concentration of 6 mM or more, or 10 mM or more.

[0054] The invention also provides proteins, such as antibodies, prepared by the methods of the invention, and methods of treating diseases, such as cancer, using these proteins (eg, antibodies).

[0055] Since nonfucosylated antibodies exhibit significantly enhanced ADCC compared to fucosylated antibodies, the antibody preparations are therapeutically superior to fucosylated antibodies and need not be completely free of fucosylated heavy chains. Residual levels of fucosylated heavy chains would not significantly inhibit the ADCC activity of a preparation with substantially nonfucosylated heavy chains. Antibodies produced in conventional CHO cells, while fully capable of adding core fucose to N-glycosylated chains, may still contain a few percent to up to 15 percent nonfucosylated antibodies. Since nonfucosylated antibodies may exhibit 10-fold higher affinity for CD16 and up to 30-100-fold enhancement of ADCC activity, even a small increase in the percentage of nonfucosylated antibodies may significantly increase the ADCC activity of the preparation. Preparations containing more nonfucosylated antibodies than produced by conventional CHO cells in culture may exhibit some degree of enhanced ADCC. Such antibody preparations are referred to herein as "reduced fucosylation" preparations. Depending on the original level of nonfucosylation obtained from conventional CHO cells, a preparation with reduced fucosylation may contain as little as 40%, 30%, 20%, 10%, or even 5% nonfucosylated antibody. Reduced fucosylation is defined functionally as a preparation that shows a 2-fold or greater enhancement in ADCC compared to antibody prepared in conventional CHO cells, and does not represent any fixed percentage of nonfucosylated species.

[0056] In other embodiments, the level of non-fucosylation is defined structurally. As used herein, a non-fucosylated antibody preparation is an antibody preparation that contains 95% or more, including 100%, non-fucosylated antibody heavy chains. A low-fucosylated antibody preparation is an antibody preparation that contains heavy chains that lack fucose, such as 50-95%, such as 75-95%, and 85-95%, of the heavy chains lack fucose. Unless otherwise indicated, low-fucosylation refers to an antibody preparation in which 50-95% of the heavy chains lack fucose, non-fucosylated refers to an antibody preparation in which 95% or more of the heavy chains lack fucose, and "low-fucosylated or non-fucosylated" refers to an antibody preparation in which 50% or more of the heavy chains lack fucose.

[0057] The level of fucosylation in an antibody preparation can be determined by any method known in the art, including but not limited to gel electrophoresis, liquid chromatography, and mass spectrometry. Unless otherwise indicated, for purposes of the present invention, the level of fucosylation in an antibody preparation is determined by hydrophilic interaction chromatography (or hydrophilic interaction liquid chromatography, HILIC), essentially as described in Example 2. To determine the level of fucosylation in an antibody preparation, the sample is denatured with PNGase F to cleave N-linked glycans and then analyzed for fucose content. LC / MS of full-length antibody chains is another method for detecting the level of fucosylation in an antibody preparation, but mass spectrometry is not inherently quantitative.

[0058] Therapeutic Uses and Methods of the Invention In some embodiments, such as the treatment of cancer or infectious diseases, it may be desirable to deplete immunosuppressive cells, such as regulatory T cells (Treg), to allow for a stronger anti-tumor or anti-infectious immune response, or to deplete the tumor-infected cells themselves. In such cases, antibodies (or antigen-binding fragments thereof) generated against cell surface proteins preferentially or exclusively expressed on immunosuppressive cells, or against cell surface proteins preferentially or exclusively expressed on tumor cells (e.g., tumor antigens) or infected cells themselves, are generated in mammalian cell lines grown in the presence of rhamnose-related fucosylation inhibitors of the invention to generate a population of hypofucosylated or nonfucosylated antibodies with enhanced ADCC activity. In other cases where pathological inflammation causes disease, such as autoimmune disorders, hypofucosylated or nonfucosylated antibodies generated in mammalian cell lines grown in the presence of rhamnose-related fucosylation inhibitors of the invention are specific for cell surface proteins preferentially or exclusively expressed on the inflammatory cells themselves.

[0059] In a preferred embodiment of the therapeutic methods of the present invention, the subject is a human.

[0060] Examples of cancers that may be treated using hypofucosylated or nonfucosylated antibodies produced by the methods of the invention include bone cancer, pancreatic cancer, skin cancer, head and neck cancer, breast cancer, lung cancer, cutaneous or intraocular malignant melanoma, kidney cancer, uterine cancer, ovarian cancer, colorectal cancer, colon cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, and the like. The cancers include cancer, soft tissue sarcoma, urethral cancer, penile cancer, hematological malignancies, childhood solid tumors, lymphocytic lymphoma, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) neoplasms, primary CNS lymphoma, tumor angiogenesis, spinal axis tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancer (including asbestos-induced cancer), metastatic cancer, and any combination of the above cancers.In a preferred embodiment, the cancer is selected from MEL, RCC, squamous cell NSCLC, non-squamous cell NSCLC, CRC, CRPC, squamous cell carcinoma of the head and neck, and esophageal, ovarian, gastrointestinal and breast cancer.The method of the present invention is also applicable to the treatment of metastatic cancer.

[0061] Other cancers include hematological malignancies including, for example, multiple myeloma, B-cell lymphoma, Hodgkin's lymphoma / primary mediastinal B-cell lymphoma, non-Hodgkin's lymphoma, acute myeloid lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia, follicular lymphoma, diffuse large B-cell lymphoma, Burkitt's lymphoma, immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, mantle cell lymphoma, acute lymphoblastic leukemia, mycosis fungoides, anaplastic large cell lymphoma, T-cell lymphoma, and precursor T-lymphoblastic lymphoma, and any combination of the above cancers.

[0062] The present invention is further illustrated by the following examples, which should not be construed as limiting. The contents of all figures and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference. EXAMPLES

[0063] Example 1 Exemplary Synthesis of Fucosylation Inhibitors Exemplary synthetic methods for preparing fucosylation inhibitors of the present invention, provided in Figures 4A and 4B, will now be described in more detail.

[0064] Step 1. [ka] A solution of compound 1 (150 g, 772 mmol, 1 equiv.), 2,2-dimethoxypropane (402 g, 3.86 mol, 473 mL, 5 equiv.), and PTSA (6.65 g, 38.6 mmol, 0.05 equiv.) in acetone (750 mL) was stirred at 20° C. for 2 h. TLC (ethyl acetate, SM (R f )=0.01, product (R f )=0.38) indicated the reaction was complete. Water (150 mL) was added to the mixture. After 30 min, PTSA was neutralized with 5% NaHCO3 solution. Acetone was removed under vacuum, and the aqueous layer was washed with petroleum ether to decompose diisopropylidene, then washed with DCM (3*200 mL). The organic layer was dried (Na2SO4) and concentrated under vacuum to give compound 2 (100 g, 55%) as an off-white solid, which was used in the next step without further purification.

[0065] Step 2. [ka] To a solution of compound 2 (100 g, 426 mmol, 1 equiv.) in DCM (700 mL) was added TEA (56.1 g, 554 mmol, 77.25 mL, 1.3 equiv.) and TosCl (105 g, 554 mmol, 1.3 equiv.). The mixture was stirred at 20° C. for 16 h.

[0066] TLC (petroleum ether:ethyl acetate=1:1, product (R f)=0.43) indicated that compound 2 was completely consumed. CH2Cl2 (200 mL) was added, and the solution was washed successively with saturated NaHCO3 (5×300 mL) and H2O (3×300 mL), dried (MgSO4), and evaporated to a syrup. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate=5 / 1 to 2 / 1) to give compound 3 (100 g, 60% yield) as a pale yellow oil.

[0067] Step 3. [ka] The two reactions are carried out in parallel. To a solution of compound 3 (45.0 g, 115 mmol, 1 equiv.) in DMSO (450 mL) under N2, cooled to 20° C., NaBH4 (21.9 g, 579 mmol, 5 equiv.) was added slowly with stirring. The mixture was stirred at 80° C. for 2 h. TLC (petroleum ether:ethyl acetate=2:1, product (R f )=0.43) indicated that compound 3 was completely consumed. The two reactions were now combined. The mixture was quenched with ice H2O (1400 mL), the mixture was stirred for 15 min, then washed with EtOAc (1000 mL), dried (Na2SO4) and evaporated. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate=5 / 1 to 2 / 1) to give compound 4 (40 g, 79% yield) as a pale yellow oil.

[0068] Step 4. [ka] To a solution of compound 4 (40 g, 183 mmol, 1 equiv.) in HO (2000 mL) was added Dowex 50H+ resin (300 g). The mixture was stirred at 80° C. for 24 h. TLC (dichloromethane:methanol=3:1, product (R f)=0.15) indicated complete consumption of compound 4. The reaction mixture was filtered and concentrated under reduced pressure to give compound 5 (30 g, crude) as a pale yellow oil.

[0069] Step 5. [ka] To a solution of compound 5 (30.0 g, 182 mmol, 1 equiv.) in Py (300 mL), DMAP (4.47 g, 36.5 mmol, 0.2 equiv.) and AcO (149 g, 1.46 mol, 136 mL, 8 equiv.) were added and the mixture was stirred at 20° C. for 12 h. TLC (petroleum ether:ethyl acetate=3:1, product (R f )=0.43) indicated that compound 5 was completely consumed. The reaction mixture was quenched by adding H2O (300 mL) and then diluted with EtOAC (500 mL). The organic layer was washed with 1N HCl (300 mL×2), dried over Na2SO4, filtered, and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate=10 / 1 to 5:1) to give compound 6 (30 g, 49% yield) as a pale yellow oil.

[0070] Step 6. [ka] Compound 6 (20.0 g, 60.1 mmol, 1 equiv.) is dissolved in DMF (110 mL). Acetic acid; hydrazine (8.31 g, 90.2 mmol, 1.5 equiv.) are added and the mixture is stirred at 25° C. under N2 for 3 h. TLC (petroleum ether:ethyl acetate=1:1, product (R f)=0.24) indicated that compound 6 was completely consumed. The reaction mixture was quenched by adding 300 mL of H2O at 0°C, then extracted with EtOAc (200 mL x 2). The organic layers were combined, washed with brine (100 mL x 3), dried over Na2SO4, filtered and concentrated under reduced pressure to give a residue. Compound 7 (12.0 g, crude) was obtained as a pale yellow oil and used in the next step without further purification.

[0071] Step 7. [ka] Compound 7 (12.0 g, 41.3 mmol, 1 equiv) was coevaporated twice with -30 mL ACN, then 50 mL ACN was added. Compound 7a (15.7 g, 45.4 mmol, 15 mL, 1.1 equiv) in 40 mL ACN was added. The mixture was cooled to 0°C. TFA.Py (1 M, 74 mL, 1.8 equiv) was added dropwise at 0-5°C. The mixture was stirred at 25°C for 1 h. Cooled to 0°C and m-CPBA (15.1 g, 74.4 mmol, 85% purity, 1.8 equiv) in 40 mL ACN was added dropwise at 0°C. The mixture was stirred at 25°C for 1 h. TLC (petroleum ether:ethyl acetate=2:1, product (R f )=0.24) indicated that compound 7 was completely consumed. Saturated Na2SO3 (400 mL) and EtOAc (600 mL) were added and the mixture was stirred at 25° C. for 20 min. The organic layer was separated, washed with Na2SO3 (300 mL*2) and brine (300 mL), dried over Na2SO4, and filtered. The residue was purified by column chromatography (SiO2, petroleum ether / ethyl acetate=2 / 1) to give compound 8 (10.0 g, 43% yield) as a pale yellow oil.

[0072] Step 8. [ka] To a solution of compound 8 (4.00 g, 7.27 mmol, 1 equiv.) in MeOH (200 mL) was added Pd / C (10%, 4.0 g), 3.63 mL of TEA under N2 atmosphere. The suspension was degassed and purged with H2 three times. The mixture was stirred at 25° C. under H2 (30 Psi) for 3 h. TLC (petroleum ether:ethyl acetate=1:1, product (R f )=0.05) indicated that compound 8 was completely consumed. The mixture was filtered through Celite, and the filter cake was washed with MeOH (30 mL) and concentrated under reduced pressure to give compound 9 (1.5 g, 55.7% yield) as a pale yellow oil.

[0073] Step 9. [ka] To a solution of compound 8 (1.5 g, 4.05 mmol, 1 eq.) was added NH3 / MeOH (7M, 70 mL, 120 eq.). The mixture was stirred at 25° C. for 12 h. LCMS (et14769-65-p1a, Rt=0.235 min) showed that the desired MS was detected. The reaction mixture was filtered and the filter cake was concentrated under reduced pressure to obtain a residue. The product was lyophilized. Compound D-Rha-phosphate (0.6 g, 61% yield) was obtained as a pale yellow oil.

[0074] Step 10. [ka] Compound 9 (0.1 g, 270 μmol, 1 eq) was coevaporated with Py (1 mL×2). Compound 9_A (98.0 mg, 135 μmol, 0.5 eq) was added and the mixture was coevaporated with Py (1 mL×2). Tetrazole (0.45 M, 1.20 mL, 2 eq) was added and the mixture was coevaporated with Py (1 mL×2). Py (2 mL) was added and degassed with N2. The mixture was stirred at 25° C. for 40 h. LCMS (et14769-78-p1D, Rt=1.157 min) showed that reactant 1 remained. Several new peaks were shown by LC-MS, and the desired compound was detected. The reaction mixture was concentrated under reduced pressure to give a residue. The residue was purified by preparative HPLC (neutral conditions). Lyophilization gave a mixture of compound 10 and compound 9 (20 mg, yield 61%) as a pale yellow oil.

[0075] Step 11. [ka] A mixture of compound 10 and compound 9 (20 mg) was dissolved in H2O (0.5 mL). A solution of MeOH / H2O / TEA (0.5 mL) was added. The mixture was stirred at 30°C for 20 minutes. LCMS (et14769-83-p1a) showed that the desired ms was detected. H2O (6 mL) was added to the resulting mixture, which was then lyophilized three times. Compound 11 and a mixture of compound 11_A (20 mg) were obtained as a pale yellow oil.

[0076] Step 12. [ka] Compound 11 and the mixture of compound 11_A (20 mg) were dissolved in Dowex 5WX8-100 (Na + The eluate was lyophilized to obtain a mixture B (15 mg) of compound GDP-D-rhamnose and compound 11 as a pale yellow solid.

[0077] Example 2 Assay for determining the proportion of nonfucosylated antibodies in a sample A preparation of nonfucosylated antibody may be analyzed to determine the percentage of afucosylated heavy chains essentially as follows.

[0078] First, the antibody is denatured with urea and then reduced with DTT (dithiothreitol). The sample is then digested with PNGase F overnight at 37° C. to remove N-linked glycans. The released glycans are collected, filtered, dried, and derivatized with 2-aminobenzoic acid (2-AA) or 2-aminobenzamide (2-AB). The resulting labeled glycans are then separated on a HILIC column, and the eluted fractions are quantified by fluorescence and dried. The fractions are then treated with an exoglycosidase, such as α(1-2,3,4,6) fucosidase (BKF), to release the core α(1,6)-linked fucose residues. Untreated and BKF-treated samples are then analyzed by liquid chromatography. Glycans containing α(1,6)-linked fucose residues show a change in elution after BKF treatment, whereas nonfucosylated glycans remain unchanged. The oligosaccharide composition is also confirmed by mass spectrometry, see, e.g., Zhu et al. (2014) MAbs 6:1474.

[0079] The percentage of nonfucosylated is calculated as 100 times the molar ratio of (glycans lacking α1,6-linked fucose to the first GlcNac residue of the N-linked glycan at N297 of the antibody heavy chain) to (the sum of all glycans at that position, which includes both glycans lacking fucose and glycans with α1,6-linked fucose). Table 7 Sequence Listing Summary [Table 2]

[0080] Equivalent: Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments disclosed herein which equivalents are intended to be encompassed by the following claims. For example, the disclosure provides the following: [Section 1] 1. A method for producing a protein with reduced fucosylation from a mammalian cell line expressing said protein, comprising: a. culturing the mammalian cell line in a medium containing a compound comprising rhamnose, and then b. isolating said protein having reduced fucosylation. A method comprising: [Section 2] Item 2. The method of item 1, wherein the isolated reduced fucosylated protein comprises at least 20% non-fucosylated protein. [Section 3] 3. The method of claim 2, wherein the isolated reduced fucosylated protein comprises at least 40% non-fucosylated protein. [Section 4] Item 4. The method according to any one of Items 1 to 3, wherein the isolated reduced fucosylated protein is hypofucosylated or nonfucosylated. [Section 5] Item 5. The method according to any one of Items 1 to 4, wherein the compound is GDP-D-rhamnose, Ac-GDP-D-rhamnose, or sodium rhamnose phosphate. [Section 6] Item 6. The method according to item 5, wherein the compound is Ac-GDP-D-rhamnose. [Section 7] Item 6. The method according to item 5, wherein the compound is GDP-D-rhamnose. [Section 8] Item 8. The method according to any one of Items 5 to 7, wherein the compound is present in the medium at 6 mM or more. [Section 9] 9. The method of claim 8, wherein the compound is present in the medium at 10 mM or more. [Section 10] Item 10. The method according to any one of items 1 to 9, wherein the compound is present in the medium substantially all the time that the mammalian cell line produces the reduced fucosylated protein. [Section 11] Item 11. The method according to any one of Items 1 to 10, wherein the protein is an antibody. [Section 12] The method of claim 11, wherein the isolated reduced fucosylation protein exhibits a 2-fold or greater enhancement in ADCC compared to the same antibody produced in the same cell line in the absence of a fucosylation inhibitor, as determined by the method described in Example 2. [Section 13] Item 13. A protein with reduced fucosylation produced by the method according to any one of items 1 to 12. [Section 14] 12. An antibody with reduced fucosylation produced by the method described in item 11. [Section 15] A method for treating cancer, comprising administering the protein of paragraph 13 to a patient in need thereof. [Section 16] A method for treating cancer, comprising administering the antibody of paragraph 14 to a patient in need thereof. [Section 17] Ac-GDP-D-rhamnose. [Section 18] D-Rhamnose phosphate.

Claims

1. 1. A method for producing a protein with reduced fucosylation from a mammalian cell line expressing said protein, comprising: a. culturing the mammalian cell line in a medium containing a compound comprising rhamnose, wherein the compound is GDP-D-rhamnose or Ac-GDP-D-rhamnose; and b. Isolating the protein with reduced fucosylation. A method comprising:

2. The method of claim 1 , wherein the isolated reduced fucosylated protein comprises at least 20% non-fucosylated protein.

3. The method of claim 2, wherein the isolated reduced fucosylated protein comprises at least 40% non-fucosylated protein.

4. The method of any one of claims 1 to 3, wherein the isolated reduced fucosylated protein is a hypofucosylated protein or a nonfucosylated protein.

5. The method according to any one of claims 1 to 4, wherein the compound is Ac-GDP-D-rhamnose.

6. The method according to any one of claims 1 to 4, wherein the compound is GDP-D-rhamnose.

7. The method of any one of claims 1 to 6, wherein the compound is present in the medium at 6 mM or more.

8. 8. The method of claim 7, wherein the compound is present in the medium at 10 mM or more.

9. The method of any one of claims 1 to 8, wherein said compound is present in the culture medium while said mammalian cell line produces said protein with reduced fucosylation.

10. The method according to any one of claims 1 to 9, wherein the protein is an antibody.

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