TW202010844A - Manufacture method of afucosylated antibodies - Google Patents

Abstract

The present disclosure is directed to methods for producing an afucosylated antibody, the afucosylated antibodies and composition thereof, and cells for producing antibodies. The method comprises introducing a nucleic acid encoding at least one modified enzyme of the fucosylation pathway to a host cell to produce the afucosylated antibody in the host cell. The disclosure method can easily, stably, and cost-effectively produce afucosylated antibodies. In addition, the afucosylated antibodies produced by the disclosed methods have increased ADCC activity and would not suppress their CDC and safety.

Description

Method for manufacturing defucosylated antibody

The present invention relates to a defucosylated protein, including defucosylated immune function molecules with enhanced activity and therapeutic properties, and a method for preparing the defucosylated protein.

Glycoproteins are involved in many basic human functions, including catalysis, signal transduction, cell-to-cell message transmission, and molecular recognition and binding. Many glycoproteins have been used for therapeutic purposes, and in the past two decades, naturally occurring recombinant forms of secreted glycoproteins have been the main products of the biotechnology industry. Examples include erythropoietin (EPO), therapeutic monoclonal antibody (therapeutic mAb), tissue plasminogen activator (tPA), and interferon-β (interferon-β , IFN-β), granulocyte-macrophage colony stimulating factor (GM-CSF) and human chorionic gonadotrophin (hCG).

There are five types of antibodies in mammals, namely IgM, IgD, IgG, IgA and IgE. Human IgG antibodies are mainly used for the diagnosis, prevention and treatment of various human diseases due to their long half-life in blood and functional characteristics, such as various effector functions. Human IgG antibodies can be further divided into the following four subclasses: IgG1, IgG2, IgG3 and IgG4. There have been many studies on antibody-dependent cellular cytotoxicity (ADCC) activity and complement-dependent cytotoxicity (CDC) as effector functions of IgG antibodies, and IgG1 Class antibodies have the largest ADCC activity and CDC activity among human IgG antibodies.

The performance of ADCC activity and CDC activity of human IgG1 subclass antibodies requires antibody Fc region and effector cells, such as killer cells, natural killer cells, activated macrophages or similar cells, and antibody receptors present on the surface of various complements ( Hereinafter referred to as "FcγR") binding. It has been shown that many amino acid residues of the second domain in the hinge region and the C region of the antibody (hereinafter referred to as "Cγ2 domain"), and the sugar chain linked to the Cγ2 region are important for this binding reaction.

Decreasing or inhibiting the fucosylation of N-polysaccharides of antibodies or Fc fusion proteins can enhance ADCC activity. ADCC is generally involved in the activation of natural killer (NK) cells, and relies on antibody-coated cells to recognize Fc receptors on the surface of NK cells. The binding of the Fc region to Fc receptors on NK cells is affected by the glycosylation state of the Fc region. In addition, the type of N-polysaccharide in the Fc region can also affect ADCC activity. Therefore, for an antibody composition or an Fc fusion protein composition, the binding affinity for FcγRIII or the ADCC activity of the composition can be increased by increasing the relative amount of defucosylated N-polysaccharide.

Many factors that can affect glycation, including species, tissues and cell types, have been shown to be very important for the way glycation occurs. In addition, the extracellular environment can directly affect glycosylation by changing culture conditions, such as serum concentration. Various methods have been proposed to change the glycosylation pattern achieved in a particular host organism, including the introduction or excessive expression of certain enzymes related to the production of oligosaccharides (U.S. Pat. No. 5,047,335; U.S. Pat. No. 5,510,261).

WO98/58964 describes antibody compositions in which substantially all N-terminally-linked oligosaccharides are G2 oligosaccharides. G2 refers to a structure with two terminals Gal and no NeuAcs (biantennary). WO99/22764 mentions antibody compositions that are substantially free of glycoproteins with N-terminally-linked G1, G0 or G-1 oligosaccharides in the CH2 domain. G1 refers to a bifurcated structure with one Gal and no NeuAcs, G0 refers to a bifurcated structure without terminal NeuAcs or Gals, and G-1 refers to one GlcNAc less in the core unit.

WO00/61739 reports that 47% of anti-hIL-5R antibodies expressed by YB2/0 (rat myeloma) cells have α1 compared to 73% of antibodies expressed by NSO (mouse myeloma) cells -6 fucose linked sugar chains. The relative ratio of fucose of αhIL-5R antibody fucose antibody expressed by different host cells is YB2/0<CHO/d<NSO.

WO02/31140 and WO03/85118 show that the modification of fucose bound to sugar chains can be controlled by using RNAi to inhibit the function of α1,6-fucosyltransferase. A method for producing an antibody composition using a cell, which includes a cell using an anti-lectin, the lectin identifying a sugar chain, wherein the first position of fucose is bound through an α-bond in a sugar chain linked to a complex N-glycoside To the 6th position of N-acetylglucosamine in the reducing end.

The structure of the sugar chain plays a very important role in the effect function of human IgG1 subclass antibodies, and by changing the structure of the sugar chain, it may be possible to prepare antibodies with stronger effect functions. However, the structure of sugar chains is diverse and complex, and the physiological role of sugar chains is still insufficient and expensive. Therefore, a method for producing defucosylated antibodies is needed.

references

1. PAULSON, James, et al., "Process for controlling intracellular glycosylation of proteins" US Patent No. 5,047,335 (1991)

2. GOOCHEE, Charles F., et al., “Method of controlling the degradation of glycoprotein oligosaccharides produced by cultured Chinese hamster ovary cells” US Patent No. 5,510,261 (1996)

3. WONG, Chi-Huey, et al., "Method and composition for synthesizing sialylated glycosyl compounds" US Patent No. 5,278,299 (1994)

4. RAJU, T., Shantha, "Methods and compositions for galactosylated glycoproteins" WO/1998/058964 (1998)

5. RAJU, T., Shantha, "Methods and compositions comprising galactosylated glycoproteins" WO/1999/022764 (1999)

6. HANAI, Nobuo, et al., "Method for controlling the activity of immunologically functional molecule" WO/2000/061739 (2000)

7. KANDA, Yutaka, et al., “cells producing antibody compositions” WO/2002/031140 (2002)

8. BLUMBERG, R. S., et al., "Central airway administration for systemic delivery of therapeutics" WO 03/077834 (2002)

9. BLUMBERG, R. S., et al., "Central airway administration for systemic delivery of therapeutics" US Patent Application Publication 2003-0235536 (2003)

10. MÖSSENER, E., et al., “Increasing the efficacy of CD20 antibody therapy through the engineering of a new type II anti-CD20 antibody with enhanced direct and immune effector cell-mediated B-cell cytotoxicity” Blood 115, 4393- 4402 (2010)

11. FERRARA, C., et al., “Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcγRIII and antibodies lacking core fucose” Proc Natl Acad Sci USA 108, 12669-12674 (2011)

The present invention relates to a novel method for producing defucosylated proteins with enhanced activity, including defucosylated antibodies. The present invention also relates to afucosylated protein produced by the method of the present invention, and cells producing afucosylated protein. The defucosylated antibodies of the present invention have increased antibody-dependent cytotoxicity (ADCC) activity compared to naturally occurring fucosylated antibodies.

One category of the invention relates to a method for producing defucosylated proteins, including defucosylated antibodies, in host cells. The method of the present invention generally includes introducing a nucleic acid encoding a modified enzyme in the fucosylation pathway into a host cell to inhibit antibody fucosylation in the host cell. The modified enzyme may be derived from enzymes in the fucosylation pathway. In some embodiments, the modified enzyme may be derived from GDP-mannonse 4,6-dehydratase (GDP-mannonse 4,6-dehydratase, GMD), GDP-4-keto-6-deoxy-D -Mannose isomerase-reductase (GDP-4-keto-6-deoxy-D-mannose epinierase-reductase, FX) and/or fucose transferase (FUT1 to FUT12, POFUT1 and POFUT2). In some embodiments, the modified enzyme may be derived from GMD or FUT. In certain embodiments, the modified enzyme may be derived from α-1,6-fucose transferase (FUT8). The modified enzyme can inhibit the function of the host cell's naturally occurring enzyme in the fucosylation pathway, thereby inhibiting the fucosylation of antibodies in the host cell.

In some embodiments, a method of producing defucosylated proteins (including defucosylated antibodies) includes (a) providing a host cell and (b) modifying a modified fucosylation pathway The nucleic acid of the enzyme is introduced into the host cell, and (c) the defucosylated protein is produced in the host cell.

Another category of the invention relates to defucosylated proteins produced by the method of the present invention, including defucosylated antibodies. Compared to naturally occurring fucosylated antibodies, defucosylated antibodies have increased and improved activity. In some embodiments, the antibody has increased and improved ADCC.

The invention also relates to cells for producing defucosylated proteins, including defucosylated antibodies.

The following examples provide the embodiments of the present invention, together with the accompanying drawings.

1 is a view of cells in RC79 (RITUXAN® performance of stable cell lines) and performance F83M, F8M1, F8M2, F8M3 F8D1 mutant cell RC79 recombinant proteins produced in the protein profiles of FUT8 or a western blot. The performance of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serves as a protein loading control group. In RC79 recombinant cells expressing mutant FUT8 enzyme, the expression level of FUT8 protein is similar to, or the same as that of parental RC79 cells.

Flow cytometric analysis showed F83M mutant RC79 RC79 recombinant cell proteins and the parental cell is the second FIG. The long dashed peak represents the RC79 recombinant cells expressing F83M stained with rhodamine-LCA. The gray filled peaks represent RC79 recombinant cells expressing F83M without Rhodamine-LCA staining (negative control group). The peak of the short dashed line represents RC79 cells stained with Rhodamine-LCA (parent cells not expressing F83M) (positive control group).

的圖。第3a及3b圖分別顯示經由來自捐贈者1(第3a圖)及捐贈者2(第3b圖)之PBMC細胞的RITUXAN®與去岩藻醣基化抗-CD20單株抗體的ADCC活性。去岩藻醣基化抗-CD20單株抗體 (R1細胞株)的ADCC活性顯著高於RITUXAN®。 Of FIG. 3a and 3b show ADCC is

Figure. Figures 3a and 3b show the ADCC activity of RITUXAN® and defucosylated anti-CD20 monoclonal antibodies via PBMC cells from donor 1 ( Figure 3a ) and donor 2 ( Figure 3b ), respectively. The ADCC activity of defucosylated anti-CD20 monoclonal antibody (R1 cell line) was significantly higher than that of RITUXAN®.

4a to 4c is a view of FIG SPR sensorgrams FcγRIIIa affinity analysis by SPR biosensor (BIACORE ^TM X100) display. Combine His-tag FcγRIIIa (1 μg/mL) and 5-80nM defucosylated anti-CD20 monoclonal antibody ( Figure 4a ), 20-320nM RITUXAN® ( Figure 4b ) or 5-80nM GAZYVA® ( section 4b ) Fig. 4c ), flow through the CM5 wafer immobilized with anti-His antibody in sequence at a flow rate of 30 μL/min. Compared to RITUXAN® and GAZYVA®, the defucosylated anti-CD20 monoclonal antibody (R1 cell line) has a stronger affinity for FcγRIIIa.

FIG 5 is a graph showing anti -CD20 CDC activity monoclonal antibodies with defucosylated RITUXAN® glycosylation. The CDC activity of the defucosylated anti-CD20 monoclonal antibody (R1 cell line) is comparable to that of RITUXAN®.

FIG. 6 is displayed in saline (vehicle), RITUXAN® or de-fucosylated anti-tumor volumes -CD20 monoclonal antibody (R1 cell line) mice Healing of FIG. Data points represent the mean ± SD of tumor volume (n=5 per group). The anti-tumor efficacy of defucosylated anti-CD20 monoclonal antibody (cell line R1) is significantly higher than that of RITUXAN®.

7 is a view of brine from to (carrier), RITUXAN® or de-fucosylated anti-tumor -CD20 mouse monoclonal antibody (R1 cell line) treating the collected weight of the FIG. The tumor weight of mice treated with defucosylated anti-CD20 monoclonal antibody (R1 cell line) was significantly lighter than that of RITUXAN® and vehicle group.

8 is shown in FIG saline (vehicle), RITUXAN® or de-fucosylation FIG -CD20 mouse monoclonal antibody anti-weight (R1 cell lines) of the treatment. The data points in the graph represent the mean ± SD of tumor volume (n=5 per group).

The present invention relates to a novel method for producing defucosylated antibodies with improved activity. The invention also relates to the antibody produced by the method of the invention and the cell producing the defucosylated antibody. Compared to naturally-occurring fucosylated antibodies, the defucosylated antibodies of the present invention have increased antibody-dependent cytotoxicity (ADCC) activity.

The following are the provided embodiments to assist those with ordinary knowledge in the technical field to which the present invention belongs to implement the present invention. Those with ordinary knowledge in the technical field to which the present invention pertains can understand that modifications or changes to the embodiments explicitly described in the present description do not deviate from the spirit or scope of the information contained in the present invention, and still belong to the scope of the present invention. The terminology used in the description is only for describing specific embodiments, and does not limit the present invention. The headings used below are for the purpose of organizing the chapters only and should not be considered as limiting the subject matter described.

All documents, patent applications, patents, drawings and other references (including parts) mentioned in the description of the present invention can be fully incorporated, as disclosed and described in the description of the invention.

Unless otherwise stated, all technical and scientific terms used in the description of the present invention have the same meaning as those generally understood in the technical field to which the present invention belongs. Unless the context indicates otherwise, the singular "a" and "the" in the description of the invention include the plural. Similarly, unless the context indicates otherwise, the word "or" is intended to include the meaning of "and". Therefore, "including A or B" means including A or B, or A and B. It should be further understood that all amino acid sizes, all molecular weights or molecular mass values of Polypeptide are approximate values and are for description only. Although methods and materials similar or identical to those described in the present invention can be used, suitable methods and materials are described below. All documents, patent specifications, patents and other documents mentioned in the description of the present invention can be incorporated into the present invention. In case of conflict, it will be restricted by the instructions (including term explanation). In addition, the materials, methods, and examples are illustrative only and are not intended to limit the invention.

1. Inhibition of rock glycosylation

One category of the invention relates to methods for inhibiting or reducing fucosylation in cells.

a. Host cell

Any suitable host cell can be used to produce defucosylated antibodies, including those derived from yeast, insects, amphibians, fish, reptiles, birds, mammals or humans, or fusion tumors. The host cell may be an unmodified cell or cell strain, or a genetically modified cell strain (eg, to promote the production of biological products). In some embodiments, the host cell is a cell line that has been modified to allow growth under desired conditions, such as in serum-free medium, in cell suspension culture, or in adherent cell culture.

The use of mammalian host cells facilitates the administration of antibodies to humans. In some embodiments, the host cell is a Chinese hamster ovary (CHO) cell, which is a cell line used to express many recombinant proteins. Additional mammalian cell lines commonly used to express recombinant proteins include 293HEK cells, HeLa cells, COS cells, NIH/3T3 cells, Jurkat cells, NSO cells, and HUVEC cells. In other embodiments, the host cell is a recombinant cell expressing antibodies.

Examples of human cell lines useful in the methods provided by the present description include 293T (embryonic kidney), 786-0 (kidney), A498 (kidney), A549 (alveolar epithelium), ACHN (kidney), BT-549 (breast ), BxPC-3 (pancreas), CAKI-1 (kidney), Capan-i (pancreas), CCRF-CEM (leukemia), COLO 205 (colon), DLD-1 (colon), DMS 114 (small cell lung) , DU145 (prostate), EKVX (non-small cell lung), HCC-2998 (colon), HCT-15 (colon), HCT-1 16 (colon), HT29 (colon), S IT-1080 (fibrosarcoma), HEK 293 (embryonic kidney), HeLa (cervical cancer), HepG2 (hepatocellular carcinoma), HL-60 (TB) (leukemia), HOP-62 (non-small cell lung), HOP-92 (non-small cell lung) , HS 578T (breast), HT-29 (colon adenocarcinoma), IG-OV1 (ovarian), IMR32 (neuroblastoma), Jurkat (T lymphocyte), K-562 (leukemia), KM 12 (colon) , KM20L2 (colon), LANS (neuroblastoma), LNCap.FGC (Caucasian prostate adenocarcinoma), LOX IMV1 (melanoma), LXFL 529 (non-small cell lung), M 14 (melanoma), M19-MEL (Melanoma), MALME-3M (melanoma), MCFIOA (breast epithelium), MCI '7 (breast), MDA-MB-453 (breast epithelium), MDA-MB-468 (breast), MDA-MB-231 (Breast), MDA-N (breast), MOLT-4 (leukemia), NCl/ADR-RES (ovary), NCI-1122.0 (non-small cell lung), NCI-H23 (non-small cell lung), NCl-H322M (Non-small cell lung), NCI-H460 (non-small cell lung), NCI-H522 (non-small cell lung), OVCAR-3 (ovary), QVCAR-4 (ovary), OVCAR-5 (ovarian), OVCAR- 8 (ovary), P388 (leukemia), P388/ADR (leukemia), PC-3 (prostate), PERC6® (El-transformed embryonic retina), RPMI-7951 (melanoma), RPMI-8226 (leukemia), RXF 393 (kidney), RXF-631 (kidney), Saos-2 (bone), SF-268 (central nervous system), SF-295 (central nervous system), SF-539 (central nervous system), SHP-77 ( Small cell lung), SH-SY5Y (neuroblastoma), SK-BR3 (breast), SK-MEL-2 (melanoma), SK-MEL-5 (melanoma), SK -MEL-28 (melanoma), SK-OV-3 (ovarian), SN12K1 (kidney), SN12C (kidney), SNB-19 (central nervous system), SNB-75 (central nervous system), SNB-78 ( Central nervous system), SR (leukemia), SW-620 (colon), T-47D (breast), THP-1 (monocyte macrophages), TK-10 (kidney), U87 (glioblastoma ), U293 (kidney), U251 (central nervous system), UACC-257 (melanoma), UACC-62 (melanoma), UO-31 (kidney), W138 (lung) and XF 498 (central nervous system) Cell line.

Examples of non-human primate cell lines useful in the methods provided by the description of the present invention include monkey kidney (CVI-76), African green monkey kidney (VERO-76), green transfected with SV40 (COS-7) Cell lines of monkey fiber cells (COS-1) and monkey kidney cells. The additional mammalian cell lines are known to those with ordinary knowledge in the technical field to which the present invention belongs, and are cataloged in the American Type Culture Collection (ATCC) catalog (Manassas, VA).

b. Modify enzymes in the fucosylation pathway

The defucosylated antibody of the present invention can be produced in a host cell, and the fucosylation pathway in this host cell has been altered in a manner that reduces or inhibits the fucosylation of the protein.

i. Modified enzyme

The phrase "modified enzyme" used in the description of the present invention refers to a protein derived from a naturally-occurring or wild-type enzyme in the fucosylation pathway, which is modified or modified to destroy the natural enzyme activity of the protein change. The modified enzyme can inhibit or interfere with its corresponding wild-type enzyme to change, inhibit or reduce the activity of the wild-type enzyme in the host cell.

Modified enzymes can be produced by changing naturally occurring enzymes, for example, by changing the total protein charge, covalently linking chemical or protein moieties, introducing amino acid substitutions, insertions and/or deletions, and/or Any combination of them. In some embodiments, the modified enzyme has amino acid substitutions, additions, and/or deletions compared to naturally occurring enzyme counterparts. In some embodiments, the modified enzyme has between 1 and about 20 amino acid substitutions, additions, and/or deletions compared to naturally occurring enzyme counterparts. The substitution, addition and insertion of amino acids can be accomplished using natural or unnatural amino acids. Non-naturally occurring amino acids include, but are not limited to, ε-N lysine, ß-alanine, ornithine, n-leucine, n-valine, hydroxyproline, thyroxine, γ-amine Butyric acid, homoserine, citrulline, aminobenzoic acid, 6-aminohexanoic acid (Aca; 6-Aminohexanoic acid), hydroxyproline, thiopropionic acid (MPA), 3-nitro-casein Amino acid, pyroglutamic acid and the like. Naturally occurring amino acids include alanine, arginine, aspartic acid, aspartic acid, cysteine, glutamic acid, glutamic acid, glycine, histidine, isoleucine , Leucine, lysine, methionine, amphetamine, proline, serine, threonine, tryptophan, tyrosine and valine.

The modified enzyme may be derived from any naturally occurring enzyme in the fucosylation pathway. For example, the modified enzyme may be derived from GDP-mannose 4,6-dehydratase (GMD), GDP-4-keto-6-deoxy-D-mannose isomerase-reductase (FX) and// Or any fucose transferase, including: 2-α-L-fucose transferase 1 (FUT1), galactosides 2-α-L-fucose transferase 2 (FUT2), galactosides 3 ( 4)-L-fucose transferase (FUT3), α(1,3) fucose transferase, bone marrow specific (FUT4), α-(1,3)-fucose transferase (FUT5), α-(1,3)-fucose transferase (FUT6), α-(1,3)-fucose transferase (FUT7), α-(1,6)-fucose transferase (FUT8) , Α-(1,3)-fucose transferase (FUT9), protein O-fucose transferase 1 (POFUT1), protein O-fucose transferase 2 (POFUT2).

In some embodiments, more than one enzyme in the fucosylation pathway is modified. In certain embodiments, the modified enzyme is derived from GMD, FX, and/or FUT8.

ii. Nucleic acid encoding modified enzyme

The defucosylated antibody of the present invention can be produced in a host cell in which the enzyme in the fucosylation pathway has been altered in a manner that reduces or inhibits the fucosylation of the protein.

In some embodiments, the host cell's fucosylation pathway is altered by introducing a nucleic acid encoding a modified enzyme in the fucosylation pathway into the host cell. For example, nucleic acid molecules encoding modified enzymes are inserted into expression vectors and transfected into host cells. The nucleic acid molecule encoding the modified enzyme can be transiently introduced into the host cell or stably fused into the host cell's genome. Standard recombinant DNA methods can be used to generate nucleic acids encoding modified enzymes, incorporate the nucleic acids into expression vectors, and introduce the vectors into host cells.

In some embodiments, the host cell may express 2 or more modified enzymes. For example, nucleic acids encoding two or more modified enzymes can be used to transfect host cells. Alternatively, more than one nucleic acid can be used to transfect the host cell, and each nucleic acid can encode one or more modified enzymes.

The nucleic acid encoding the modified enzyme may contain additional nucleic acid sequences. For example, the nucleic acid may contain protein tags, selection markers, or regulatory sequences that control the expression of proteins in the host cell, such as promoters, enhancers, or other expression control fragments that control the transcription or translation of nucleic acids (eg, polyadenylation signals). Such regulatory sequences are already known. Those of ordinary skill in the technical field to which the invention pertains can understand that the expression vector, including the selection of regulatory fragments, can be selected based on several factors, including the selection of the host cell to be transformed, the amount of protein expression required, and so on. Exemplary regulatory sequences for mammalian host cell expression, including viral fragments in mammalian cells that direct high protein expression, for example, derived from cytomegalovirus (CMV) (eg, CMV promoter and/or enhanced ), simian virus 40 (Simian Virus, SV40) (for example, SV40 promoter and/or enhancer), adenovirus (for example, adenovirus major late promoter (AdMLP)) and polyoma virus Promoter and/or enhancer.

In certain embodiments, nucleic acid sequences containing modified enzymes derived from GMD, FX, and/or FUT are introduced into host cells. In host cells that express modified enzymes, the fucosylation pathway is altered, inhibited, or reduced.

c. Host cells expressing modified enzymes

Another category of the invention relates to host cells that express modified enzymes in the fucosylation pathway. The performance of the modified enzyme in the host cell interferes with the activity of the wild-type enzyme, resulting in inhibition or reduction of the fucosylation pathway. Therefore, proteins (eg, antibodies) produced in the host fine cells expressing modified enzymes are defucosylated (non-fucosylated).

The phrase "low-fucosylated cell" or "low-fucosylated host cell" as used in the present invention refers to the fact that since the cell expresses a modified enzyme in the fucosylation pathway, fucose Cells whose basification pathway has been inhibited or reduced.

Low-fucosylated cells can be prepared by transfecting a expression vector containing a nucleic acid sequence encoding a modified enzyme in the fucosylation pathway into a host cell. Transfection can be performed using techniques known in the art. For example, chemical-based methods (eg, lipids, calcium phosphate, cationic polymers, DEAE-dextran, activated dendrimers, magnetic beads, etc.), and instrument-based methods (eg , Electroporation, biojet technology, microinjection, laser/light injection, etc.), or by virus-based methods for transfection.

The screening markers present on the expression vector can be used to screen and isolate transfected cells from untransfected cells. In addition, transfected cells with inhibited or reduced fucosylation pathways can be screened and isolated from cells with normal fucosylation pathways by various techniques. For example, antibodies, lectins, metabolic markers, or chemical enzyme strategies can be used to determine fucosylation. In addition, cells with inhibited or reduced fucosylation pathways can be screened by exposing transfected cells to lentil lectin (Lens culinaris agglutinin, LCA, Vector laboratories L-1040). LCA recognizes the α-1,6-fucosylated trimannose core structure of N-linked oligosaccharides and allows cells expressing this structure to enter the cell death pathway. Therefore, cells that survive after exposure to LCA have an inhibited or reduced fucosylation pathway and are considered low-fucosylated cells.

2. Defucosylated protein

Another category of the invention relates to a method for producing defucosylated proteins. In some embodiments, the defucosylated protein is a defucosylated antibody.

a. protein

Non-limiting examples of proteins that can be produced as defucosylated proteins include GP-73, thrombin, HBsAg, hepatitis B virus particles, α-acid-glycoprotein, α-1-chymotrypsin, α -1-chymotrypsin, α-1-chymotrypsin histidine-proline-less (His-Pro-less), α-1-antitrypsin, serum transferrin (Serotransferrin), plasma ceruloplasmin , Α-2-macroglobulin, α-2-HS-glycoprotein, α-fetoprotein, binding globin, fibrinogen γ chain precursor, immunoglobulin (including IgG, IgA, IgM, IgD, IgE And similar), APO-D, prokinin, histidine-rich glycoprotein, complement factor 1 precursor, complement factor I heavy chain, complement factor I light chain, complement C1s, complement factor B precursor, complement Factor B Ba fragment, complement factor B Bb fragment, complement C3 precursor, complement C3 β chain, complement C3 α chain, C3a allergic toxin, complement, C3b α′ chain, complement C3c fragment, complement C3dg fragment, complement C3g fragment, complement C3d fragment, complement C3f fragment, complement C5, complement C5 β chain, complement C5 α chain, C5a allergic toxin, complement C5α′ chain, complement C7, α-1 B glycoprotein, B-2-glycoprotein, vitamin D-binding Protein, Inter-α-trypsin inhibitor heavy chain H2, α-1B-glycoprotein, angiotensinogen precursor, angiotensin-1, angiotensin-2, angiotensin-3, GARP protein, β -2-glycoprotein, Clusterin (Apo J), integrin α-8 precursor glycoprotein, integrin α-8 heavy chain, integrin α-8 light chain, hepatitis C virus particles, elf- 5. Kininogen, HSP33-homologous, lysinyl endopeptidase and protein 32 precursor rich in repeated leucine.

b. Antibody

The term "antibody" used in the description of the present invention broadly covers intact antibodies and fragments thereof that can be fucosylated. For example, antibodies include fully assembled immunoglobulins (eg, multiple strains, single strains, monospecific, multispecific, chimeric, deimmunized, humanized, human, primate, single chain, single domain, (Synthetic and recombinant antibodies); parts of intact antibodies with desired activity or function (eg, immunological fragments of antibodies that contain Fab, Fab', F(ab')2, Fv, scFv, single-domain fragments); and contain Peptides and proteins of the Fc domain that can be fucosylated (eg, Fc-fusion proteins).

The term "defucosylated antibody" as used in the description of the present invention refers to an antibody produced under conditions in which fucosylation is inhibited or significantly reduced compared to antibodies produced under natural conditions or Fragment. The defucosylated antibody produced by the method of the present invention may be fully (100%) defucosylated, or may include a mixture of fucosylated and defucosylated molecules. For example, in some embodiments, the antibodies produced by the methods of the present invention may contain about 20% to about 100% of afucosylated molecules. In other embodiments, the antibody produced by the method of the present invention may contain about 40% to about 100% of afucosylated molecules. In certain embodiments, the antibodies produced by the methods of the invention may contain about at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97, 98%, 99% or 100% of fucosylated molecules. Not all N-glycated antibodies or fragments thereof (eg, Fc-fusion proteins) need to be defucosylated.

b. Antibody type

Any antibody can be produced as a defucosylated antibody using the method of the present invention. There is no limitation on the types of antibodies that can be produced using the method of the present invention. The following is a non-exhaustive list of antibodies that can be produced.

Examples of antibodies that recognize tumor-associated antigens include anti-GD2 antibodies, anti-GD3 antibodies, anti-GM2 antibodies, anti-HER2 antibodies, anti-CD52 antibodies, anti-MAGE antibodies, anti-HM124 antibodies, anti-parathyroid glands Hormone-related protein (PTHrP) antibody, anti-basic fibroblast growth factor antibody and anti-FGF8 antibody, anti-basic fibroblast growth factor receptor antibody and anti-FGFS receptor antibody, anti- Insulin-like growth factor antibodies, anti-insulin-like growth factor receptor antibodies, anti-PMSA antibodies, anti-vascular endothelial cell growth factor antibodies, anti-vascular endothelial cell growth factor receptor antibodies and the like.

Examples of antibodies that recognize antigens associated with allergy or inflammation include anti-IL-6 antibodies, anti-IL-6 receptor antibodies, anti-IL-5 antibodies, anti-IL-5 receptor antibodies and anti-IL-4 antibodies, Anti-tumor necrosis factor antibody, anti-tumor necrosis factor receptor antibody, anti-CCR4 antibody, anti-chemokine antibody, anti-chemokine receptor antibody and the like.

Examples of antibodies that recognize antigens associated with circulating organ diseases include anti-GpIIb/IIIa antibodies, anti-platelet-derived growth factor antibodies, anti-platelet-derived growth factor receptor antibodies, and anti-blood coagulation factor antibodies and the like.

Examples of antibodies that recognize antigens associated with viral or bacterial infections include anti-gpl 20 antibodies, anti-CD4 antibodies, anti-CCR4 antibodies, and anti-verotoxin antibodies and the like.

Many therapeutic antibodies are commercially available, for example, binding VEGF (eg, Bevacizumab (AVASTIN®)), EGFR (eg, Cetuximab (ERBITUX®)), HER2 (eg, Trastuzumab (HERCEPTIN®)) and CD20 (eg , Rituximab (RITUXAN®)), and TNFa (eg, Etanecept (ENBREL®), which includes the receptor binding domain of the TNF receptor (p75)), CD2 (eg, Alefacept (AMEVIVE®), which contains LFA -3 CD2-binding region), or B7 (Abatacept (ORENCIA®), which includes CTLA4's B7-binding region) antibody Fc-fusion protein.

3. Method for preparing defucosylated protein

Defucosylated proteins of the invention are produced in hypofucosylated cells, which include defucosylated antibodies. Using techniques known in the art, such as transfection of low-fucosylated cells with expression vectors encoding proteins, defucosylated proteins can be expressed in low-fucosylated cells.

Techniques known in the art can be used to prepare expression vectors encoding proteins. For example, to reverse transcribe an amino acid sequence into a nucleic acid sequence to construct an expression vector, it is preferable to use a nucleic acid codon optimized for the organism in which the protein is expressed. Then, the nucleic acid encoding the protein and any regulatory fragments thereof are assembled and inserted into the desired expression vector. The expression vector may contain additional nucleic acid sequences, such as protein tags, screening markers, or regulatory sequences that control protein expression, as described above for expression vectors containing modified enzymes. The expression vector can then be introduced into the host cell using transfection. Transfection can be performed using techniques known in the art. For example, chemical-based methods (eg, lipids, calcium phosphate, cationic polymers, DEAE-dextran, activated dendrimers, magnetic beads, etc.) can be used, with instrument-based methods (eg , Electroporation, biojet technology, microinjection, laser/light injection, etc.), or transfection by virus-based methods. The protein can then be expressed in the transfected cells under conditions suitable for the chosen expression system and host. The expressed protein can then be purified using affinity columns or other techniques known in the art.

Transfect the nucleic acid encoding the modified enzyme (to be a low-fucosylated cell) and the nucleic acid encoding the protein (to express this protein) in any order into the host cell to produce defucosylated protein. For example, a nucleic acid encoding a modified enzyme (to be a hypofucosylated cell) can be transfected into a host cell, and then a nucleic acid encoding a protein (to express this protein) can be transfected. Alternatively, the nucleic acid encoding the protein (to express this protein) can be transfected into the host cell first, and then the nucleic acid encoding the modified enzyme (to be a low-fucosylated cell) can be transfected. In another variation, the nucleic acid encoding the modified enzyme (to be a hypofucosylated cell) and the nucleic acid encoding the protein (to express this protein) can be transfected into the host cell at the same time.

In a specific embodiment, according to the following steps, by preparing low-fucosylated cells first, and then transfecting the protein-encoding nucleic acid into the low-fucosylated cells to produce defucosylated proteins : A) obtain a host cell suitable for protein expression; b) transfect the nucleic acid encoding the modified enzyme into this host cell; c) screen and/or isolate transfusified cells with low fucosylation; d) Transfection of protein-encoding nucleic acid into low-fucosylated cells; e) screening and/or isolation of low-fucosylated cells transfected with protein-encoding nucleic acid; f) induction of proteins in low-fucosylated Performance in cells.

In another embodiment, according to the following steps, by transfecting the nucleic acid encoding the protein into the host cell, and then transfecting the cell with the nucleic acid encoding the modified enzyme, the defucosylated protein is produced: a) obtain a host cell suitable for protein expression; b) transfect the nucleic acid encoding the protein into the host cell; c) screen and/or isolate cells transfected with the nucleic acid encoding the protein; d) encode the modified enzyme Transfected nucleic acid into cells of step (c); e) screening and/or isolation of transfected cells with low fucosylation in step (d); f) induced protein in low fucosylated cells Performance.

In a variation of the above embodiment, the defucosylated protein can be produced as follows: a) obtaining a host cell that expresses or overexpresses the protein; b) transfects the nucleic acid encoding the modified enzyme into this cell; c) Screen and/or isolate transfected cells with low fucosylation; d) Induce protein performance in low fucosylated cells.

In yet another embodiment, as follows, by simultaneously transfecting a nucleic acid encoding a modified enzyme (to be a hypofucosylated cell) and a nucleic acid encoding a protein (to express this protein) into a host cell, to To produce afucosylated protein: a) obtain a host cell suitable for expressing a protein; b) transfect the first nucleic acid encoding the protein and the second nucleic acid encoding the modified enzyme into the host cell; c) screen and/or Or isolate transfected cells that express protein and have low fucosylation; d) Induce protein performance in low fucosylated cells.

The afucosylated proteins, including antibodies, produced using the method of the present invention described above can be purified by methods known in the art. For example, the defucosylated proteins, including antibodies, produced by the method of the present invention can be purified by physiochemical fractionation, antibody-specific affinity, and antigen-specific affinity.

4. Improved properties of defucosylated antibodies

Compared to antibodies produced using standard methods, the defucosylated antibodies produced by the method of the present invention have improved properties.

The activity of defucosylated antibodies can be measured by ELISA, fluorescence method and the like. The cytotoxic activity of antigen-positive cultured cell lines can be evaluated by measuring their ADCC and CDC. Appropriate models of animal species that are relatively close to humans can be used to assess the safety and therapeutic effects of antibodies in humans.

a. Increased ADCC activity

Compared to antibodies produced using standard methods, the defucosylated antibodies of the present invention have increased ADCC activity.

"ADCC activity" used in the method of the present invention refers to the ability of an antibody to cause an antibody-dependent cytotoxicity (ADCC) response. ADCC is a cell-mediated response in which antigen-specific cytotoxic cells that express FcRs (eg, natural killer (NK) cells, neutrophils, and macrophages) recognize antibodies that bind to the surface of target cells, which then cause the target cells Decomposition (ie, kill). The main mediator cells in ADCC are natural killer (NK) cells. NK cells express FcyRIII, where FcyRIIIA is an activated receptor and FcyRIIIB is an inhibitory receptor. Monocytes express FcγRI, FcγRII and FcγRIII. In vitro assays, such as the assay described in Example 3, can be used to directly assess ADCC activity.

In vitro assays can be used to directly assess ADCC activity. In some embodiments, the ADCC activity of the defucosylated antibody of the present invention is at least 0.5, 1, 2, 3, 5, 10, 20, 50, 100 times its wild-type control antibody.

Since the defucosylated antibody has increased ADCC activity, the defucosylated therapeutic antibody can be administered in a lower amount or concentration than its fucosylated antibody. In some embodiments, the concentration of the afucosylated antibody of the invention may be at least 2, 3, 5, 10, 20, 30, 50, or 100 times lower than the concentration of its fucosylated antibody. In some embodiments, the defucosylated antibody of the present invention can exhibit a higher maximum target cell lysis than its wild-type counterpart. For example, the maximum target cell breakdown of the defucosylated antibody of the present invention is 10%, 15%, 20%, 25%, 30%, 40 higher than the maximum target cell breakdown of its wild-type antibody %, 50% or more.

b. Increased CDC activity

Compared to antibodies produced using standard methods, the defucosylated antibodies of the invention have increased complement dependent cytotoxicity (CDC) activity.

The "CDC activity" used in the method of the present invention refers to the reaction of one or more components in the complement system that recognizes the antibody bound to the target cell and then decomposes the target cell. The defucosylated antibody of the present invention does not reduce or inhibit CDC activity, but on the contrary, it maintains CDC activity similar to or higher than that of its fucosylated counterpart.

The present invention further provides defucosylated antibodies with enhanced CDC function. In one embodiment, the Fc variants of the invention have increased CDC activity. In another embodiment, the defucosylated antibody has at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 50-fold, or at least 100-fold higher than the relative molecule CDC activity.

4. Use defucosylated antibodies

It can be intravenously (iv), subcutaneously (sc), intra-muscularly (im), intradermal (id), intraperitoneal (ip) or via any mucosal surface, such as oral ( Orally, po), sublingually (sl), cheek, nose, rectum, vagina, or via the pulmonary route to administer the defucosylated antibody of the present invention.

Defucosylated antibodies are useful for the treatment or prevention of various diseases, including cancer, inflammatory diseases, immune and autoimmune diseases, allergies, circulatory organ diseases (eg, arteriosclerosis), and viral or bacterial infections.

The dosage of the defucosylated antibody of the present invention will vary depending on the subject to be administered and the specific mode. The required dose can be changed according to many factors well known to those of ordinary skill in the technical field to which the invention belongs, including but not limited to, antibody target, type of subject, and size/weight of the subject. The dose may be 0.1 to 100,000 μg/kg body weight. Defucosylated antibodies can be administered in single or multiple doses. The defucosylated antibody can be administered once in a 24-hour period, multiple times during a 24-hour period, or continuous injection. Defucosylated antibodies can be administered continuously or on a specific schedule. The effective dose can be inferred from the dose-response curve obtained from the animal model.

4. Specific embodiments

Specific embodiments of the invention include, but are not limited to the following:

(1) A method for producing afucosylated protein in a host cell, comprising introducing at least one nucleic acid encoding a modified enzyme of the fucosylation pathway into the host cell to produce Fucosylated protein.

(2) The method of (1), wherein the modified enzyme is derived from GDP-mannose 4,6-dehydrogenase (GMD), GDP-4-keto-6 -Deoxy-D-mannose isomerase-reductase (GDP-4-keto-6-deoxy-D-mannose epinierase-reductase, FX), or fucosyltransferase (FUT).

(3) The method of (1), wherein the modified enzyme is derived from fucose transferase (FUT).

(4) The method of (1), wherein the modified enzyme is derived from α-1,6-fucose transferase (FUT8).

(5) The method of (1), wherein the modified enzyme reduces or inhibits the activity of the wild-type enzyme derived from the modified enzyme in the host cell.

(6) The method of (1), wherein the modified enzyme inhibits or reduces fucosylation in the host cell.

(7) The method according to (1), wherein the defucosylated protein produced in the host cell is a defucosylated antibody or a fragment thereof.

(8) The method of (7), wherein the defucosylated antibody or fragment thereof is at least 90% defucosylated.

(9) The method of (7), wherein the defucosylated antibody has increased antibody-dependent cellular cytotoxicity (ADCC) activity compared to its fucosylated antibody.

(10) The method of (7), wherein the complement-dependent cytotoxicity (CDC) of the defucosylated antibody is not reduced or inhibited compared to its fucosylated counterpart.

(11) A method for producing afucosylated protein in a host cell, comprising a first nucleic acid sequence encoding a modified enzyme of the fucosylation pathway, and a second nucleic acid sequence encoding the protein to be produced Transfected into host cells.

(12) The method of (11), wherein the host cell transfects the first nucleic acid sequence and the second nucleic acid sequence simultaneously.

(13) The method according to (12), wherein the defucosylated protein produced is an antibody and/or a fragment thereof.

(14) The method according to (11), wherein the first nucleic acid encodes a GDP-mannose 4,6-dehydratase (GMD), GDP-4-keto-6-deoxy-D-mannose isomerase -Modified enzymes of reductase (FX) and/or fucose transferase (FUT).

(15) The method according to (11), wherein the first nucleic acid encodes a modified enzyme derived from α-1,6-fucose transferase (FUT8).

(16) The method according to (11), including the following steps: a) transfecting the first nucleic acid encoding the modified enzyme into the host cell; b) screening and isolating the low-fucosylated transfected cells; c ) Transfect the second nucleic acid encoding the protein to be produced into the hypofucosylated cell; d) express the protein to be produced in the low fucosylated cell.

(17) The method according to (11), wherein the defucosylated protein produced is an antibody and/or a fragment thereof.

(18) The method according to (11), including the following steps: a) transfecting a second nucleic acid encoding the protein to be produced into a host cell; b) screening and isolating cells transfected with the second nucleic acid; c) transcoding The first nucleic acid of the modified enzyme is transfected into the cell in step (b); d) Screening and isolation of transfusified cells with low fucosylation; e) Demonstration of production in this low fucosylated cell Of protein.

(19) The method according to (18), wherein the defucosylated protein produced is an antibody and/or a fragment thereof.

5. Additional embodiments

Additional embodiments of the invention include, but are not limited to the following:

(1) A method for producing defucosylated antibodies, comprising: introducing a nucleic acid encoding at least one modified enzyme into a host cell to produce defucosylated antibodies in the host cell.

(2) The method of (1), wherein the modified enzyme is derived from GDP-mannose 4,6-dehydratase (GMD), GDP-4-keto-6-deoxy-D-mannose isomerase -Reductase (FX), or fucose transferase (FUT).

(3) The method of (1), wherein the modified enzyme is derived from fucose transferase (FUT).

(4) The method of (1), wherein the modified enzyme is derived from α-1,6-fucose transferase (FUT8).

(5) The method of (1), wherein the modified enzyme inhibits the activity of wild-type fucosylated enzyme in the host cell.

(6) The method of (1), wherein the modified enzyme inhibits and/or reduces fucosylation of the antibody in the host cell.

(7) The method of (1), wherein the defucosylated antibody has increased ADCC.

(8) The method of (1), wherein the CDC activity of the defucosylated antibody is not reduced or inhibited.

(9) A method for producing defucosylated antibodies, comprising: a) providing a host cell, b) introducing a nucleic acid encoding at least one modified enzyme into the host cell, and c) producing de-rock in the host cell Fucosylated antibody.

(10) The method of (9), wherein in step (a), the host cell includes at least one nucleic acid encoding an antibody.

(11) As in the method of (9), the nucleic acid encoding the antibody is further introduced into the host cell after step (b).

(12) The method of (9), wherein the modified enzyme is derived from GDP-mannose 4,6-dehydratase (GMD), GDP-4-keto-6-deoxy-D-mannose isomerase -Reductase (FX), and/or fucose transferase (FUT).

(13) The method of (9), wherein the modified enzyme is derived from fucose transferase (FUT).

(14) The method of (9), wherein the modified enzyme is derived from α-1,6-fucose transferase (FUT8).

(15) The method of (9), wherein the modified enzyme inhibits the activity of fucosylase in the host cell.

(16) The method of (9), wherein the modified enzyme inhibits and reduces the glycosylation of the antibody in the host cell.

(17) The method of (9), wherein the defucosylated antibody has increased ADCC.

(18) The method of (9), wherein the CDC activity of the defucosylated antibody is not reduced or inhibited.

(19) A defucosylated antibody produced by the method of (1) or (9), wherein the defucosylated antibody has increased ADCC activity.

(20) The method according to (19), wherein the defucosylated antibody is a human antibody or a fragment thereof.

(21) The method according to (19), wherein the defucosylated antibody maintains the original CDC activity.

(22) A pharmaceutical composition comprising the defucosylated antibody of (19) and a pharmaceutically acceptable carrier or excipient.

(23) A cell with no or low fucosylation, including a nucleic acid encoding at least one modified enzyme.

Additional specific embodiments of the present invention include, but are not limited to the following embodiments.

[Example 1]

Preparation of modified enzyme in the fucosylation pathway and stable cell lines expressing the modified enzyme

1. Cell line

Commercially available CHOdhfr(-) cell line (ATCC CRL-9096), which is a mutant strain of CHO cells lacking dihydrofolate reductase activity, purchased from Culture Collection and Research Center (CCRC), Taiwan ). The CHOdhfr ^(-) cell line was divided into 3 separate cultures and treated as follows: The first culture was transfected with a expression vector encoding RITUXAN® (Rituximab, a chimeric monoclonal antibody against protein CD20). A stable cell line expressing RITUXAN® was obtained and identified as RC79.

The second culture was transfected encoding HERCEPTIN® (Trastuzumab, a monoclonal antibody against the protein HER2). A stable cell line expressing HERCEPTIN® was obtained and identified as HC59.

The third culture was untreated cells and was maintained as CHOdhfr(-) cell line.

2. Construction of expression vector encoding modified enzymes of FUT8 and GMD

Constructed many expression vectors encoding modified enzymes FUT8 and GMD.

The F83M, F8M1, F8M2, F8M3 and F8D1 mutants represent different modifications to α-1,6-fucose transferase and wild-type FUT8 protein (GenBank No. NP_058589.2), respectively. Table 1 summarizes the modifications made to the wild-type nucleic acid sequence for each FUT8 vector and the amino acid changes produced in the enzymes shown. In particular, F83M represents a mutant with three modifications in R365A, D409A and D453A in the wild-type FUT8 protein. F8M1, F8M2, and F8M3 respectively represent mutants with a modification in K369E, D409K, and S469V in the wild-type FUT8 protein. F8D1 represents a mutant with deletion of amino acid residues at positions 365 to 386 in the wild-type FUT8 protein.

Table 2 summarizes the modifications made by the GMD vector to the wild-type nucleic acid sequence, and the amino acid changes produced in the enzymes represented. In particular, the mutant GMD4M represents a modification of GDP-mannose 4,6-dehydratase, the wild-type GMD protein (GenBank No. NP_001233625.1, which has 4 mutations in the wild-type GMD protein, in T155A, E157A , Y179A and K183A.

All nucleic acid sequences encoding F83M, F8M1, F8M2, F8M3, F8D1, and GMD4M were synthesized by GeneDireX and sub-selected into the pHD expression vector (pcDNA3.1Hygro, Invitrogen, Carlsbad, CA, cat.no.V870-20, with dhfr Gene) PacI/EcoRv or BamHI/EcoRV positions to form pHD/F83M, pHD/F8M1, pHD/F8M2, pHD/F8M3, pHD/F8D1 and pHD/GMD4M plastids.

3. Preparation of stable recombinant cell lines expressing modified enzymes

Transfection of pHD/F83M, pHD/F8M1, pHD/F8M2, pHD/F8M3, pHD/F8D1 and pHD/GMD4M plastids by electroporation (PA4000 PULSEAGILE® electroporator, Cyto Pulse Sciences) into different cell lines, including ( a) RC79 cell line (CHO cell expressing RITUXAN®), (b) HC59 cell line (CHO cell expressing HERCEPTIN®), and (c) CHOdhfr ^(-) cell (CHO cell lacking dihydrofolate reductase activity mutation) Strain).

a. RC79 cells

The transfected RC79 cell line was first cultivated in RC79 medium containing 0.1 to 0.25 mg/mL Hygromycin (containing 0.4 μM MTX, 0.5 mg/mL Geneticin, 0.05 mg/mL bleomycin) (Zeocin), 4 mM Glutamax-I and 0.01% F-68 in EX-CELL® 302 serum-free medium). Next, the transfected cells were cultured in EX containing 0.4 μM MTX, 0.5 mg/mL geneticin, 0.05 mg/mL bleomycin, 4 mM Glutamax-I, 0.01% F-68 and 0.25 mg/mL hygromycin -CELL®302 serum-free medium and separated with Lens culinaris agglutinin (LCA) as described below to produce 5 cell pools, including RC79F83M, RC79F8M1, RC79F8M2, RC79F8M3, RC79F8D1 and RC79-GMD4M cell line.

b. HC59 cells

The transfected HC59 cell line was first cultivated in HC59 medium containing 0.1 to 0.25 mg/mL hygromycin (containing 0.8 μM MTX, 0.5 mg/mL geneticin, 0.05 mg/mL bleomycin and 4 mM Glutamax-I EX-CELL® 325 PF CHO medium). Next, the transfected cells were cultured in EX-CELL® 325 PF containing 0.8 μM MTX, 0.5 mg/mL geneticin, 0.05 mg/mL bleomycin, 4 mM Glutamax-I and 0.25 mg/mL hygromycin In CHO medium and as described below, it was isolated with LCA to produce a cell bank of HC59F83M cell line.

c. CHOdhfr ^(-) cell

CHOdhfr ^(-) cells were first cultured in EX-CELL® 325 PF CHO medium containing 4 mM Glutamax-I and 0.1 to 0.25 mg/mL hygromycin. Next, the transfected cells were cultured in EX-CELL® 325 PF CHO medium containing 4 mM Glutamax-I, 0.25 mg/mL hygromycin and 0.01 μM MTX to generate a cell bank of C109F83M cell line.

4. Isolation of cells with low fucosylation

In this example, Rhodamine-labeled Lens culinaris agglutinin (LCA) (Vector Laboratories, Cat. RL-1042) was used to select cells with low fucosylation.

All RC79, HC59 and CHO transfected strains were subjected to the initial selection medium containing hygromycin as the selection pressure, and then the core structure of α-1,6-fucosylated trimannose, which can identify N-linked oligosaccharides, The cells that express this structure are sent to the LCA of the cell death pathway for final screening. First to 1.2 x 10 ⁵ cells / mL, the RC79, HC59 or CHO transfected lines were seeded in 2.5mL fresh media containing 0.4mg / mL LCA, the survival rate and the cells were counted on day 3 or 4. The cells were cultured in this preliminary screening medium until the cell survival rate reached 80%. After the cell survival rate reached 80%, the cells were resuspended in fresh screening medium containing gradient concentrations of LCA at 1.2 x 10 ⁵ cells/mL. The LCA screening is repeated several times until the final LCA concentration reaches 0.6 to 1.2 mg/mL.

To analyze the amount of fucose on the cell surface, cells were labeled with LCA and analyzed by flow cytometry. First, the cells were planted in complete medium without LCA for 14 days to remove signal interference from the screening reagent LCA. Subsequently, 1mL ice-cold PBS at 3 x 10 ⁵ cells were washed twice, and resuspended in 1% bovine serum albumin and 5μg / mL LCA in 200μl of ice-cold PBS. After incubating on ice for 30 minutes, the cells were washed twice with 1 mL of ice-cold PBS. The cells were resuspended in 350 μl cold PBS and analyzed using a FACScalibur ^™ flow cytometer (BD Biosciences, San Jose, CA).

Next, 1 x 10 ⁷ cells were washed twice with 10 mL of ice-cold PBS, and resuspended in 6.5 mL of ice-cold PBS containing 1% bovine serum albumin and 5 μg/mL LCA. After incubating on ice for 30 minutes, the cells were washed twice with 10 mL of cold PBS. The cells were resuspended in 1 mL of ice-cold PBS containing 1% heat-inactivated fetal bovine serum (GIBCO, Cat. 10091-148) and antibiotic-antimycotic agent (Invitrogen, Cat. 15240062).

Cells were analyzed and sorted with FACSAria ^™ or Influx ^™ Cell Sorter (BD Biosciences, San Jose, CA). For different cell lines, 1-3 classifications are required to produce a homogeneous population with low fucosylated cells. In addition, the CLONEPIX ^™ 2 system (MOLECULAR DEVICES®) was used to isolate stable cell lines with low fucosylation and moved to 96-well plates. After culturing for about 2 weeks, the cells were transferred to a 6-well plate and analyzed by flow cytometry. Then, the cells with low fucosylation were moved to a filter tube for fed-batch culture to evaluate the cell performance and the degree of fucosylation of antibodies purified from the resulting cells.

5. Preparation of C109F83M cell line expressing RITUXAN®

After isolating the low-fucosylated CHOdhfr ^(-) cells (C109F83M cells) with LCA, the nucleic acid encoding RITUXAN® was transfected into the cells by electroporation (PA4000 PULSEAGILE® electroporator, Cyto Pulse Sciences). A low-fucose single cell line of C109F83M, AF97, was isolated and transfected with nucleic acid encoding RITUXAN® to express RITUXAN® by electroporation. The transfected strain was transferred to a 25T flask flask containing non-selective medium to resume growth. After 48 hours, the transfected strain was cultured in a selection medium containing 4 mM GlutaMAX-I, hygromycin-B, bleomycin and 0.01 μM MTX. Single cells were selected using the CLONEPIX™ 2 system to produce AF97 anti-CD20 cell line.

The obtained cells are low-fucosylated CHOdhfr ^(-) cells expressing RITUXAN®, and are referred to herein as AF97 anti-CD20 cell lines.

[Example 2] Performance and analysis of defucosylated antibody 1. Antibody performance and purification

The cells with low fucosylation activity obtained in Example 1 were cultured in batches or fed batches to express antibodies. The antibody purified from the cells is subjected to monosaccharide analysis to quantitatively analyze the sugar chain in the Fc region.

The recombinant RC79 cells were cultured in EX-CELL® 302 serum-free medium containing 4 mM Glutamax and 0.01% F-68, and maintained in a shaking incubator (Infors Multitron Pro) at 37° C. and 5% CO ₂ .

Recombinant HC59 cells were cultured in EX-CELL® 325 PF CHO medium containing 0.8 μM MTX, 0.5 mg/mL geneticin, 0.05 mg/mL bleomycin, 4 mM Glutamax-I and 0.25 mg/mL hygromycin And maintained in a shaking incubator (Infors Multitron Pro) at 37°C, 5% CO ₂ .

Cell culture parameters are routinely monitored daily. A hemocytometer was used to exclude trypan blue to determine cell density and survival rate. When the cell survival rate is less than 60%, the conditioned medium is collected by centrifugation and the expressed antibody is purified with protein A resin. The protein A column was equilibrated with 5 column volumes of 0.1M Tris, pH 8.3, and then the sample was added to the column. Unbound protein was washed away with 0.1M Tris, pH 8.3 (2 column volumes) and PBS, pH 6.5 (10 column volumes). The column was further washed with 0.1 M sodium acetate, pH 6.5 (10 times the column volume). Finally, the antibody was eluted with 0.1 M glycine, pH 2.8, and neutralized with the same elution volume of 0.1 M Tris, pH 8.3.

2. Judgment of antibody N-polysaccharide map analysis

The N-polysaccharide map was analyzed with the ACQUITY UPLC® system. First, at 37°C, 0.3 mg of antibody sample was digested with 3 U PNGase-F in 0.3 mL of digestion buffer (15 mM Tris-HCl, pH 7.0) for 18 hours. The released N-polysaccharide was separated from the antibody by ultrafiltration using an AMICON® Ultra-0.5mL 30K device at 13,000 rpm for 5 minutes, and then freeze-dried for 3 hours. Next, the dried N-polysaccharide was dissolved in 30 μL of ddH ₂ O and 45 μL of 2-AB labeling reagent (DMSO-acetic acid containing 0.34M aminobenzamide and 1M sodium cyanoborohydride (7:3 v/v ) In the solvent) and incubate at 65°C for 3 hours. Remove excess 2-AB labeling reagent in PD MINITRAP ^™ G10 size exclusion column. The labeled N-polysaccharide was freeze-dried overnight and redissolved in 50 μL ddH ₂ O for UPLC detection. At 60°C, the N-glycan pattern was obtained by ACQUITYUPLC® system and Glycan BEH amide column. Different forms of N-glycans were separated with a linear gradient of 100 mM ammonium formate, pH 4.5/acetonitrile.

The results of flow cytometry showed that LCA binding was extremely low on the cell surface overexpressing F83M protein in all cell types. Similarly, LCA binding was not detected on RC79 cells overexpressing F8M1, F8M2, F8M3, F8D1 or GMD4M proteins (data not shown).

Table 3 shows the antibodies produced in RC79 and HC59 cells with unmodified fucosylation pathway and RC79 and HC59 cells whose fucosylation pathway is modified by overexpressing F83M modified enzyme N-glycan map. The data in Table 3 shows that the anti-CD20 and anti-ErbB2 antibodies produced in cells with unmodified fucosylation pathways are mostly heavily fucosylated. In particular, in these cells, only 3.67% of anti-CD20 and 3.64% of anti-ErbB2 antibodies were defucosylated. In contrast, antibodies produced in cells that overexpress F83M modified enzymes have a very low degree of fucosylation. In particular, in cells that overexpress F83M modified enzymes, approximately 98.86-98.91% of anti-CD20 and approximately 92.12-96.52% of anti-ErbB2 antibodies are defucosylated.

In addition, Table 4 shows RC79 cells with unmodified fucosylation pathway and RC79 cells with modified fucosylation pathway by overexpressing one of the modified enzymes of F8M1, F8M2, F8M3, F8D1 or GMD4M N-glycan profile of the antibody produced in The data in Table 4 shows that the anti-CD20 antibody produced in RC79 cells with an unmodified fucosylation pathway is mostly heavily fucosylated. In particular, in these cells, only 3.67% of the anti-CD20 antibody was defucosylated. In contrast, antibodies produced in RC79 cells that over-express modified enzymes have a very low degree of fucosylation. In particular, the degree of defucosylation of anti-CD20 antibodies produced by cells that overexpress F8M1, F8M2, F8M3, F8D1, or GMD4M modified enzymes is about 92.78% to about 97.16%, as shown in Table 4 . .

Table 4 also shows that the degree of defucosylation of antibodies produced by cells that overexpress one of FUT8 modified enzymes (F8M1, F8M2, F8M3, F8D1) is 95.70 to 97.16%, and is modified by overexpression of GMD The antibody produced by the enzyme (GMD4M) has a degree of defucosylation of 92.78%. These results confirm that the antibodies produced in cells that over-express FUT8 modified enzymes are more de-glycosylated than those produced in cells that over-express GMD mutant proteins.

The results in Tables 3 and 4 confirm that host cells that have been designed to express antibodies can be transfected with vectors that express modified enzymes (FUT8 or GMD) in the fucosylation pathway. This result also shows that the antibodies produced in these transfected cells are defucosylated antibodies.

In addition, the degree of fucosylation of antibodies produced in the AF97 cell line was evaluated. The results in Table 5 show that the antibodies produced in AF97 cells that overexpress F83M modified enzymes have a very low degree of fucosylation. In particular, 97.83% of the anti-CD20 antibodies produced in AF97 cell lines were defucosylated. In comparison, commercially available RITUXAN® (MABTHERA®) has a degree of defucosylation of 3.92%.

The results in Table 5 show that defucosylated antibodies can be produced in cells that were first transfected with nucleic acids encoding modified enzymes and then transfected with nucleic acids encoding antibodies a second time. Therefore, the method of the present invention can be used to modify cells to produce defucosylated antibodies.

3. Expression of FUT8 protein in recombinant cells

Decompose RC79 cells and recombinant cells expressing FUT8 modified enzymes (eg, F8M1, F8M2, F8M3, or F8D1) into 1% Triton X containing phosphatase inhibitor cocktail (Sigma-Aldrich, Cat. S8820) -100. DC ^TM (cleaner) protein analysis (BIO-RAD) reagent was used to confirm the protein concentration in the supernatant of decomposed cells. Each sample containing 30 μg of protein supernatant was separated and transferred to nitrocellulose by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) On the plain film. By using 25mM Tris-HCl (pH7.4) containing 120mM NaCl, 0.1% gelatin (w/w) and 0.1% TWEEN® 20 (polyethylene glycol sorbitan monolaurate (v/w), The nitrocellulose membrane was blocked for 1 hour and incubated with anti-FUT8 antibody (Abcam, Cat.ab204124, 1:500) and GAPDH antibody (GeneTex, Cat.GT239, 1:10000) at 4°C overnight. The membrane was washed 3 times with 25 mM Tris-HCl (pH 7.4) (containing 120 mM NaCl, 0.1% gelatin (w/w) and 0.1% TWEEN® 20 (v/w)) for 5 minutes, then at room temperature with Goat anti-rabbit IgG (Jackson ImmunoResearch, Cat. 111-035-144) and goat anti-mouse IgG HRP (GeneTex, Cat. GTX213111-01, 1:10000) were reacted for 1 hour respectively. After additional washing, use SIGMAFAST DAB with Metal Enhancer (Sigma, Cat. D0426) to analyze the membrane.

FIG 1 is a western blot showing expression of FUT8 protein source parent cells RC79 and RC79 recombinant cell in the performance of the modified enzyme. The expression levels of FUT8 protein in recombinant cells and RC79 parent are similar. These results indicate that the production of defucosylated antibodies in RC79 cells expressing modified enzymes is independent of the amount of FUT8 protein expressed. These results show that FUT8 modified enzymes can interfere with wild-type FUT8 protein in cells to inhibit and/or reduce the fucosylation pathway, allowing recombinant cells to efficiently produce defucosylated antibodies.

Compared with other methods that rely on inhibiting or down-regulating the wild-type FUT8 gene, or using RNAi to reduce FUT8 protein expression, the mechanism for producing defucosylated antibodies using the method of the present invention is novel and unique.

4. Stability of recombinant cells

The stability of RC79 recombinant cells expressing F83M modified enzyme was evaluated.

The RC79 recombinant cells were cultured in medium without screening reagents for 3 months. Weekly flow cytometry analysis was used to monitor the fucosylation of the cells, and the ACQUITY UPLC® System and Glycan BEH Amide columns were used monthly to determine the composition of the purified antibody N-glycans for 3 consecutive Month, as described above. The non-binding properties of LCA were maintained during the 90-day evaluation period, indicating that the fucosylation pathway was inhibited and/or reduced throughout the study ( Figure 2 ).

In addition, during the 72-day period, the degree of defucosylation of anti-CD20 antibodies produced in 5 stable RC79F83M cell lines (R4-R8) was evaluated. As shown in Table 6 , all RC79F83M cell lines produced highly defucosylated antibodies during the 72-day study.

These results of this study prove that the recombinant cell line expressing the modified enzyme prepared by the method of the present invention is stable and produces highly defucosylated antibodies for a long time.

[Example 3] ADCC activity of defucosylated antibody

In order to evaluate the in vitro cytotoxicity of the purified anti-CD20 antibody obtained in Example 2, the ADCC activity was measured according to the following method.

1. Preparation of effector cell solution

Add human peripheral blood (100 mL) from healthy donors to a VACUTAINER® test tube containing heparin sodium. The whole blood sample was diluted 1:1 with RPMI 1640 serum-free (SF) medium and mixed gently. Using Ficoll-Paque PLUS, monocytes were isolated by slowly adding 24 mL of diluted blood to Ficoll-Paque and centrifuging at 400 xg for 32 minutes at 25°C. The buffy coat was fully dispensed into two 50 mL centrifuge tubes containing 20 mL RPMI 1640 medium, and then mixed twice. Next, the mixture was centrifuged at 1,200 rpm for 12 minutes at 25°C to obtain a supernatant. RPMI 1640 SF medium (13 mL) was added to the supernatant to resuspend PBMC cells. The cells were centrifuged at 1,200 rpm for 12 minutes at 25°C to obtain a supernatant. RPMI medium (10 mL) was added to the supernatant to resuspend PBMC cells. A sufficient volume of PBMC cell suspension was added to the 75T flask, and each flask had approximately 15 mL of cells, and its final cell density was 1.5 x ¹⁰⁶ cells/mL. IL-2 (2.5 μg/mL) was added to all flasks at a final concentration of 3 ng/mL. PBMC cells were cultured in a 37°C, 5% CO ₂ incubator for 18 hours. PBMC cells stimulated with IL-2 were collected and centrifuged at 1,200 rpm for 12 minutes at 25°C, and then the supernatant was discarded. PBS (10 mL) was added and mixed with the cells. The cells were centrifuged at 1,200 rpm for 12 minutes at 25°C to remove the supernatant. The cells were resuspended at RPMI AM, and the final concentration was adjusted to 2×10 ⁷ cells/mL.

2. Preparation of target cell solution

The cell suspension from the 75T flask was centrifuged at 1,000 rpm for 5 minutes, the supernatant was removed, and then washed with 10 mL of 1X PBS. The washed cells were centrifuged at 1,200 rpm for 5 minutes, and the supernatant was removed. In RPMI assay medium, the cells were resuspended to prepare a target cell solution was ⁵ x 10 5 cells / mL. The target cell solution (40 L of ⁵ x 10 5 cells / mL) were added to a V-bottom 96-well culture plate holes cells. Next, 20 μL of the prepared commercially available RITUXAN® solution (MABTHERA®) (25-0.0025 μg/mL) (positive control group) and defucosylated antibody (R1 cell line) solution (25-0.0025 μg) /mL), or RPMI analysis medium (negative control group) was added to the hole, and mixed with the target cell solution. The V-bottom 96-well cell culture plate was cultured in a 37°C, 5% CO ₂ incubator for 30 to 60 minutes.

3. ADCC activity analysis

The effector cell solution (40 L of 8 x 10 ⁵ effector cells / hole) or 40 L of RPMI was added to the culture dish analysis, to the reaction solution to a target cell. Centrifuge the plate at 300 xg for 4 minutes. The plate was reacted at 37°C with 5% CO ₂ for 4 hours. Before collecting the supernatant, the lysis solution (10 μL) of CYTOTOX 96® was added to the trays of the Tmax and BlkV group, and reacted for 1 hour. The V-bottom 96-well cell culture dish was centrifuged at 300 xg for 4 minutes, and 50 μL of supernatant was transferred from the 96-well cell culture dish to the hole of the flat-bottom analysis dish.

Lactate dehydrogenase (LDH) (2 μL) was added to 10 mL of LDH positive control diluent to prepare LDH positive control solution. The prepared LDH positive control solution (50 [mu]L) was added to the hole of the 96-well flat bottom analysis dish.

LDH reconstituted matrix mixture (50 μL) was added to each test hole of the analytical disk. Cover the plate and incubate in the dark at room temperature for 30 minutes. Stop solution (50 μL) was added to each test hole of the plate. Immediately after adding the stop solution, the absorbance at a wavelength of 490 nm was recorded. Using each group (S, PBMC, T, E, and Tmax) blank removal absorbance values, ADCC activity was calculated according to the following formula.

Where S is the absorbance value of the LDH released by the sample (target cell + PBMC + anti-CD20 antibody); PBMC is the absorbance value of the LDH released by the target cell and PBMC; E is the absorbance value of the LDH released by the PBMC; T is the spontaneity of the target cell The absorbance value released by sexual LDH; and Tmax is the absorbance value of the maximum LDH released by the target cell.

Compared to the commercially available RITUXAN® (MABTHERA®), the defucosylated anti-CD20 antibody (R1 cell line) was found in PBMC from both donor 1 ( Figure 3a ) and donor 2 ( Figure 3b ) A significantly stronger and higher ADCC response is induced in the cells.

As shown in Table 7 , the EC _{50 of the} defucosylated anti-CD20 antibody from the RC79F83MR1 cell line was significantly lower than that of the commercially available RITUXAN®, which is a fucosylated anti-CD20 antibody. In particular, the defucosylated anti-CD20 antibody (R1 cell line) had an EC ₅₀ of 1.7 ng/mL and 4.6 ng/mL in PBMC cells from donors 1 and 2, respectively. In contrast, fucosylated anti -CD20 antibody (MABTHERA®) each having EC 18.2ng / mL and 35.0ng / mL in the ₅₀ PBMC cells from donor 1 and 2 of the.

These results of this study demonstrate that the defucosylated anti-CD20 antibody (R1 cell line) exhibited ADCC activity 7.68 to 10.7 times that of the fucosylated anti-CD20 antibody (MABTHERA®).

[Example 4] Binding affinity of defucosylated antibodies

Use an immobilization wizard coupled to the anti-histidine (anti-His) antibody of BIACORE® CM5 chip with amine coupling kit and BIACORE® X100 control software to evaluate fucosylation and de-rocking Binding affinity of fucosylated anti-CD20 antibody to His-tagged FcγRIIIa recombinant protein.

At a rate of 10 μL/min, His-labeled FcγRIIIa recombinant protein (1 μg/mL) was injected onto the anti-His antibody-immobilized CM5 wafer for 20 seconds.

Defucosylated anti-CD20 antibodies (5, 10, 20, 40, and 80 nM) from cell line 1 and commercially available fucosylated anti-CD20 antibodies RITUXAN® (MABTHERA®) (20, 40, 80, 160, and 320 nM) and commercially available defucosylated anti-CD20 antibody GAZYVA® (obinutuzumab) (5, 10, 20, 40, or 80 nM) were injected through the wafer at a flow rate of 30 μL/min for 3 minutes, respectively. The electrophoresis buffer flows through the wafer at a flow rate of 30 μL/min for 5 minutes. Glycine, pH 1.5 (10 mM) was injected into the wafer at a flow rate of 30 μL/min for 60 seconds.

Use BIACORE® X100 evaluation software to analyze the stress map of each cycle to obtain the values of equilibrium dissociation constant (K _D ), binding rate constant (Ka) and dissociation rate constant (Kd). The stress map of each cycle is adapted with 1:1 Langmuir combined with the model. If the Chi ² value is lower than 1/10X Rmax value, then the appropriate adaptation model and kinetic binding parameters are credible.

Figures 4a to 4c show typical SPR response patterns of the three antibodies tested. A typical SPR response profile shows that the conditions used for this analysis (eg, binding time, dissociation time, and antibody concentration range) are appropriate. In addition, the Chi ² value of the three antibodies is lower than 1/10X Rmax value, indicating that the 1:1 Langmuir model is suitable for the adaptation of the response map of all three antibodies.

As shown in Table 8, to fucosylated anti -CD20 antibody (R1 cell line) binding affinity for the FcγRIIIa 10 times stronger than MABTHERA® (K _D R1 cell line = 13.0nM, MABTHERA® = 151.5nM) . In addition, the defucosylated anti-CD20 antibody (R1 cell line) has a binding affinity to FcγRIIIa that is three times stronger than GAZYVA® (K _D = 13.0 nM for R1 cell line, GAZYVA® = 39.9 nM).

These results of this study confirm that the afucosylated anti-CD20 antibody (R1 cell line) prepared according to the present invention has a higher price than the commercially available fucosylated anti-CD20 antibody RITUXAN® (MABTHERA®) and the commercially available Fucosylated anti-CD20 antibody (GAZYVA®) has stronger FcγRIIIa binding affinity.

[Example 5] CDC activity of defucosylated antibody

The CDC activity of the defucosylated antibody produced by the method of the present invention is evaluated.

Daudi cells were cultured in RPMI medium, and when the cell density reached 1×10 ⁶ cells/mL, subculture was carried out (subculture density: 2-3×10 ⁵ cells/mL). Daudi cells were collected and centrifuged at 300 rpm for 5 minutes. Cells were resuspended in RPMI medium to prepare a concentration of 1 x 10 ⁵ cells / mL cell suspension. After resuspension, seed 100 μL of cell suspension or 100 μL of RPMI medium into the holes of a white 96-well dish.

The commercially available RITUXAN® (MABTHERA®) and defucosylated anti-CD20 antibody (R1 cell line) were prepared in physiological saline at a concentration of 120 μg/mL to 0.234 μg/mL. Next, 25 μL of RITUXAN® (MABTHERA®) or defucosylated anti-CD20 antibody (R1 cell line) at a concentration of 120 μg/mL to 0.234 μg/mL was added to white 96 wells containing Daudi cells or RPMI medium Holes in the plate. Add CELLTITER-GLO® reagent (20 μL) to each well and then mix. The plate was placed on a microplate shaker and shaken at 750 rpm for 2 minutes, and then incubated in the dark at room temperature for 10 minutes. A multi-mode reader with a high-sensitivity fluorescent box was inserted to detect the luminous intensity (integration time: 1 second) to calculate the EC ₅₀ value of the anti-CD20 antibody and the relevant CDC activity of the antibody.

Figure 5 shows a comparison of CDC activity of defucosylated anti-CD20 antibody (R1 cell line) and RITUXAN®. The EC ₅₀ value of the defucosylated anti-CD20 antibody (R1 cell line) was 0.682 μg/mL, which was higher than the EC ₅₀ value of RITUXAN® (EC ₅₀ = 0.582 μg/mL).

GAZYVA® is a commercially available defucosylated anti-CD20 antibody that has been shown to induce ADCC activity but inhibit CDC activity (E. Mössener et al. (2010); C. Ferrara et al. (2011)). The results obtained from other studies indicate that in order to achieve efficient cancer treatment, the amount of GAZYVA® should be increased. In contrast, the results of this example and Example 5 demonstrate that the defucosylated anti-CD20 antibody produced by the method of the present invention induces ADCC activity and maintains CDC activity similar to its fucosylated antibody . Therefore, the defucosylated anti-CD20 antibody of the present invention performs better than GAZYVA®.

[Example 6] Evidence of the efficacy of defucosylated antibodies in animal models

In this example, a B-cell lymphoma subcutaneous xenograft model was used to demonstrate the anti-tumor efficacy of the defucosylated antibody of the present invention. SU-DHL-4 is a B-cell lymphoma cell line that displays a large amount of CD20 on the cell membrane and can grow and form solid tumors under the skin. Therefore, a xenograft model in SCID/Beige mice was developed to compare the antitumor efficacy of the defucosylated antibody (R1 cell line) with the commercially available RITUXAN® (MABTHERA®).

SU-DHL-4 cells were cultured in flasks with RPMI medium (CM). When the cell concentration of 0.8-1.0 x 10 ⁶ cells / mL, the cell suspension was collected and centrifuged at 300g for 5 minutes to remove the supernatant. Resuspend the cells in a new medium containing some conditioned medium (fresh CM: conditioned CM=9:1). Cells at ratio of 1: 2 to 1:10 (number of cells seeded: obtaining the total number of cells) subcultured and the cells at a concentration of at least 1 x 10 ⁵ cells / mL. The culture plate was cultured at 37°C. SU-DHL-4 cells were cultured in 5 150T flasks. When the cell concentration reached 0.8 to 1.0 x 10 ⁶ cells/mL, the cell suspension was collected into a 50 mL test tube, and then centrifuged at 1,200 rpm for 5 minutes to remove the supernatant. Using serum-free RPMI medium, adjust the cell concentration to 1 x 10 ⁸ cells/mL. On ice, use a pre-cooled syringe with an 18G needle and mix the cell suspension with an equal volume of MATRIGEL® in a 50 mL test tube. The final cell concentration is 5 x ¹⁰⁷ cells/mL. Using a pre-chilled 1 mL syringe with a 23G *1” needle, 5 x 10 ⁷ cells/mL Matrigel-SU-DHL-4 cell mixture (100 uL) was injected subcutaneously into the back of each mouse (SCID/Beige mice) The right side of the area. The total number of inoculated cells is 5 x 10 ⁶ cells. The tumor volume of each mouse is measured using calipers every 3 or 4 days, and is calculated by the equation: V=0.5 x ab ² , where a and b represent respectively The length and width of the tumor.

When the tumor volume reached about 200 mm ³ (198.25±55.53 mm ³ ), it occurred about 20 days after tumor inoculation, and the mice were divided into three groups of 5 mice each, followed by saline (vehicle), commercially available RITUXAN® (MABTHERA) or afucosylated anti-CD20 antibody (R1 cell line) treatment. Mice were injected with 0.2 mL of 0.1 mg/mL antibody weekly for 3 weeks. Twice a week, the weight and tumor size of all mice were measured with electronic scales and digital calipers. At the end of the treatment period, the mice were sacrificed, tumor tissue was removed, and weighed. Then, 10% formalin buffer was used to fix the tumor tissue at room temperature for subsequent experiments.

As shown in Figure 6 , the defucosylated anti-CD20 antibody (R1 cell line) showed significantly stronger anti-tumor efficacy than RITUXAN®. There was a statistically significant difference in tumor volume between the vehicle group and the defucosylated anti-CD20 antibody (R1 cell line) treatment group (P<0.001, tested by Student's t). In contrast, RITUXAN® did not have statistically significant differences in tumor volume compared to the vehicle-only group. At a dose of 1 mg/kg, defucosylated anti-CD20 antibody (R1 cell line) inhibited tumor growth more effectively than RITUXAN® (R1 cell line=468±148mm ³ ; RITUXAN®=1407±241mm ³ ) , As shown in Table 9 . There was a statistically significant difference in tumor volume between the defucosylated anti-CD20 antibody (R1 cell line) and the RITUXAN® group (P<0.001).

The tumor weight of the defucosylated anti-CD20 antibody (R1 cell line) treatment group was significantly smaller than that of the vehicle-only group (P<0.001), as shown in Figure 7 . However, at the same dose, RITUXAN® did not show a statistically significant difference in tumor weight compared to the vehicle group. Therefore, compared with the RITUXAN® group, in terms of tumor weight, the defucosylated anti-CD20 antibody (R1 cell line) was significantly reduced (R1 cell line=0.27±0.15g; RITUXAN®=0.62±0.09g, P <0.01), as shown in Table 9 . Defucosylated anti-CD20 antibody (R1 cell line) inhibits tumor growth more effectively than RITUXAN®, which can also correspond to the results of tumor volume.

During the treatment period, the weight of mice in all groups gradually increased, as shown in Figure 8 .

The results of these studies indicate that the defucosylated anti-CD20 antibody (R1 cell line) is safe.

<110> Lianya Pharmaceutical United Biopharma Peng Wenjun Chen Huirong

<120> Method for producing defucosylated antibody

<130> 2018.XX.XX

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<170> PatentIn version 3.5

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Claims (19)

A method for producing a defucosylated protein in a host cell, comprising introducing at least one nucleic acid encoding a modified enzyme of the fucosylation pathway into the host cell to produce the Defucosylated protein. The method as described in item 1 of the patent application scope, wherein the modified enzyme is derived from GDP-mannose 4,6-dehydratase (GDP-mannose 4,6-dehydratase, GMD), GDP-4-keto -6-deoxy-D-mannose isomerase-reductase (GDP-4-keto-6-deoxy-D-mannose epinierase-reductase, FX), or fucosyltransferase (FUT). The method as described in item 1 of the patent application scope, wherein the modified enzyme is derived from fucose transferase (FUT). The method as described in item 1 of the patent application scope, wherein the modified enzyme is derived from α-1,6-fucose transferase (FUT8). The method as described in item 1 of the patent application scope, wherein the modified enzyme reduces or inhibits the activity of the wild-type enzyme from which the modified enzyme is derived in the host cell. The method as described in item 1 of the patent application scope, wherein the modified enzyme inhibits or reduces fucosylation in the host cell. The method according to item 1 of the patent application scope, wherein the defucosylated protein produced in the host cell is a defucosylated antibody or fragment thereof. The method according to item 7 of the patent application scope, wherein the defucosylated antibody or fragment thereof is at least 90% defucosylated. The method as described in item 7 of the patent application range, wherein the defucosylated antibody has increased antibody-dependent cellular cytotoxicity (ADCC) compared to its fucosylated antibody active. The method as described in item 7 of the patent application scope, wherein the complement dependent cytotoxicity (CDC) of the defucosylated antibody is not reduced compared to its fucosylated counterpart inhibition. A method for producing a defucosylated protein in a host cell, comprising a first nucleic acid sequence encoding a modified enzyme of one of the fucosylation pathways, and a second nucleic acid encoding a protein to be produced The sequence is transfected into the host cell. The method according to item 11 of the patent application scope, wherein the host cell transfects the first nucleic acid sequence and the second nucleic acid sequence simultaneously. The method as described in item 12 of the patent application scope, wherein the defucosylated protein produced is an antibody or a fragment thereof. The method as described in item 11 of the patent application scope, wherein the first nucleic acid encoding is derived from GDP-mannose 4,6-dehydratase (GMD), GDP-4-keto-6-deoxy-D-mannose A modified enzyme of isomerase-reductase (FX) and/or fucose transferase (FUT). The method according to item 11 of the patent application scope, wherein the first nucleic acid encodes a modified enzyme derived from α-1,6-fucose transferase (FUT8). The method as described in item 11 of the patent application scope includes the following steps: a) transfecting the first nucleic acid encoding the modified enzyme into the host cell; b) screening and isolating the low-fucosylated transfection Staining the cell; c) transfecting the second nucleic acid encoding the protein to be produced into the low-fucosylated cell; d) expressing the protein to be produced in the low-fucosylated cell. The method according to item 16 of the patent application scope, wherein the defucosylated protein produced is an antibody and/or a fragment thereof. The method as described in item 11 of the patent application scope includes the following steps: a) transfecting the second nucleic acid encoding the desired protein into the host cell; b) screening and isolating the transfected second nucleic acid Cells; c) transfect the first nucleic acid encoding the modified enzyme to the cells in step (b); d) select and isolate low-fucosylated transfected cells; e) at the low The fucosylated cells express the protein to be produced. The method as described in item 18 of the patent application scope, wherein the produced defucosylated protein is an antibody and/or a fragment thereof.

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