TWI748124B - 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 functional molecules with enhanced activity and therapeutic properties, and a method for preparing the defucosylated protein.

Glycoproteins are involved in many basic functions of humans, including catalysis, signal transduction, message transmission between cells, and molecular recognition and binding. Many glycoproteins have been used for therapeutic purposes, and in the past two decades, the recombinant form of naturally occurring secreted glycoproteins has been the main product of the biotechnology industry. Examples include erythropoietin (EPO), therapeutic monoclonal antibody (therapeutic monoclonal antibody, therapeutic mAb), tissue plasminogen activator (tPA), interferon-β (interferon-β) , IFN-β), granulocyte-macrophage colony stimulating factor (granulocyte-macrophage colony stimulating factor, GM-CSF) and human chorionic gonadotrophin (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 in the diagnosis, prevention and treatment of various human diseases due to their long half-life in the 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 the effector function of IgG antibodies, and IgG1 subtypes have been reported. The class of antibodies has the largest ADCC activity and CDC activity among human IgG antibodies.

The performance of ADCC activity and CDC activity of human IgG1 subclass antibody requires antibody Fc region and effector cells, such as killer cells, natural killer cells, activated macrophages or similar cells and antibody receptors 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 antibody hinge region and C region (hereinafter referred to as "Cγ2 domain"), and sugar chains linked to the Cγ2 region are important for this binding reaction.

Reducing or inhibiting the fucosylation of the N-polysaccharide of the antibody or Fc fusion protein can enhance ADCC activity. ADCC generally participates in the activation of natural killer (NK) cells and relies on the recognition of antibody-coated cells by Fc receptors on the surface of NK cells. The binding of the Fc region to the Fc receptor on NK cells will be affected by the glycosylation status of the Fc region. In addition, the type of N-polysaccharide in the Fc region also affects ADCC activity. Therefore, for an antibody composition or an Fc fusion protein composition, the relative amount of the defucosylated N-polysaccharide can be increased to increase the binding affinity to FcγRIII, or the ADCC activity of the composition.

Many factors that can affect glycation, including species, tissues, and cell types, have all been shown to be very important to how 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 patterns achieved in specific host organisms, including the introduction or overexpression of certain enzymes involved in 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-terminal-linked oligosaccharides are G2 oligosaccharides. G2 means that it has two terminal Gals and does not have the biantannary structure of NeuAcs. WO99/22764 mentions substantially no antibody composition of glycoproteins with N-terminally-linked G1, G0 or G-1 oligosaccharides in the CH2 domain. G1 refers to a bifurcation structure with one Gal and no NeuAcs, G0 refers to a bifurcation structure without terminal NeuAcs or Gals, and G-1 refers to one GlcNAc less than the core unit.

WO00/61739 reported that, compared with 73% of antibodies expressed by NSO (mouse myeloma) cells, 47% of anti-hIL-5R antibodies expressed by YB2/0 (rat myeloma) cells had α1 -6 sugar chain linked by fucose. 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 RNAi can be used to inhibit the function of α1,6-fucosyltransferase to control the modification of fucose bound to sugar chains. A method of using cells to produce an antibody composition, which includes the use of anti-lectin cells, the lectin recognizes the sugar chain, and the first position of fucose is combined through the α-bond in the sugar chain linked by the compound 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 effector function of human IgG1 subclass antibodies, and by changing the structure of the sugar chain, it may be possible to prepare antibodies with stronger effector functions. However, the structure of sugar chains is diverse and complex, and solving the physiological role of sugar chains is still insufficient and expensive. Therefore, there is a need for a method of producing afucosylated antibodies.

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 the defucosylated protein produced by the method of the present invention and the cell that produces the defucosylated protein. Compared with naturally occurring fucosylated antibodies, the defucosylated antibodies of the present invention have increased antibody-dependent cellular cytotoxicity (ADCC) activity.

One category of the present 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 the fucosylation of the antibody in the host cell. The modified enzyme can be derived from an enzyme in the fucosylation pathway. In certain 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 a specific embodiment, the modified enzyme may be derived from α-1,6-fucose transferase (FUT8). The modified enzyme can inhibit the function of the enzyme naturally present in the host cell's fucosylation pathway, thereby inhibiting the fucosylation of the antibody in the host cell.

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

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

The present invention also relates to cells that produce defucosylated proteins, including defucosylated antibodies.

The embodiments of the present invention are provided in the following examples, 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. In RC79 recombinant cells expressing mutant FUT8 enzyme, the expression level of FUT8 protein is similar to or the same as the expression level of FUT8 protein in parental RC79 cells.

Flow cytometric analysis showed F83M mutant RC79 RC79 recombinant cell proteins and the parental cell is the second FIG. The peak of the long dashed line represents the F83M-expressing RC79 recombinant cells stained with rhodamine-LCA. The gray-filled peaks represent F83M-expressing RC79 recombinant cells (negative control group) that have not been stained with rhodamine-LCA. The peak of the short dashed line represents RC79 cells stained with rhodamine-LCA (parental cells that do not express 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 the defucosylated anti-CD20 monoclonal antibody (R1 cell line) is 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. The His-tag FcγRIIIa (1μg/mL) and 5-80nM defucosylated anti-CD20 monoclonal antibody (Figure 4a) , 20-320nM RITUXAN® (Figure 4b) or 5-80nM GAZYVA® (Figure 4a) Figure 4c) , flow through the anti-His antibody-immobilized CM5 chip in sequence at a flow rate of 30 μL/min. Compared with 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 equivalent 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 the 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 RITUXAN® and the vector 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 figure represent the mean ± SD of body weight (n=5 per group).

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

The following implementations are provided 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 belongs can understand that the modifications or changes of the embodiments clearly described in the description of the present invention will 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 terms used in the description are only used to describe specific embodiments and do not limit the present invention. The headings used below are only for the purpose of organizing the chapters and should not be seen as limiting the subject described.

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

Unless otherwise specified, all technical and scientific terms used in the description of the present invention have the same meaning as those commonly understood by those with ordinary knowledge in the technical field to which the present invention belongs. Unless the context indicates otherwise, the singular "one" and "the" in the description of the invention include the plural meaning. Similarly, unless the context dictates 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 understood that all the amino acid sizes, all molecular weights or molecular mass values of the multipeptides are approximate values and are for description only. Although methods and materials similar or identical to those described in the description of 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 the event of a conflict, it will be subject to the restrictions of this release (including the explanation of terms). In addition, the materials, methods, and examples are merely illustrative in nature, and are not intended to limit the present invention.

1. Inhibition of rock glycosylation in cells

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

a. Host cell

Any suitable host cell can be used to produce aglycosylated antibody, including host cells derived from yeast, insects, amphibians, fish, reptiles, birds, mammals or humans, or fusion tumors. The host cell may be an unmodified cell or cell line, or a genetically modified cell line (for example, to promote the production of biological products). In some embodiments, the host cell is a cell strain that has been modified to allow growth under desired conditions, such as in a 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 generally 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 in the description of this invention include 293T (embryo 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 (fetal 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 (Ovary), OVCAR- 8 (ovary), P388 (leukemia), P388/ADR (leukemia), PC-3 (prostate), PERC6® (El-deformed 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 (ovary), 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 in the description of the present invention include monkey kidney (CVI-76) transfected with SV40 (COS-7), African green monkey kidney (VERO-76), green A cell line 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. Modification of enzymes in the fucosylation pathway

The afucosylated antibody of the present invention can be produced in a host cell, and the fucosylation pathway in the host cell has been altered in a way 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 naturally occurring or wild-type enzymes in the fucosylation pathway, which is modified or destroyed in a way that changes or destroys 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, covalent linkage chemistry or protein moiety, introducing amino acid substitutions, insertions and/or deletions, and/or Any combination of it. 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 non-natural amino acids. Non-naturally occurring amino acids include, but are not limited to, ε-N lysine, ß-alanine, ornithine, leucine, norvaline, hydroxyproline, thyroxine, and γ-amine Butyric acid, homoserine, citrulline, aminobenzoic acid, 6-aminohexanoic acid (Aca; 6-Aminohexanoic acid), hydroxyproline, thiol propionic acid (MPA), 3-nitro-tyrosine Amino acid, pyroglutamate and the like. Naturally occurring amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, and isoleucine , Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine and Valine.

The modified enzyme can be derived from any naturally occurring enzyme in the fucosylation pathway. For example, the modified enzyme can 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), galactoside 2-α-L-fucose transferase 2 (FUT2), galactoside 3 ( 4)-L-Fucosyltransferase (FUT3), α(1,3)Fucosyltransferase, bone marrow-specific (FUT4), α-(1,3)-Fucosyltransferase (FUT5), α-(1,3)-Fucosyltransferase (FUT6), α-(1,3)-Fucosyltransferase (FUT7), α-(1,6)-Fucosyltransferase (FUT8) , Α-(1,3)-Fucosyltransferase (FUT9), Protein O-Fucosyltransferase 1 (POFUT1), Protein O-Fucosyltransferase 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 afucosylated 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 way that reduces or inhibits the fucosylation of the protein.

In some embodiments, the fucosylation pathway of the host cell is changed by introducing a nucleic acid encoding a modified enzyme in the fucosylation pathway into the host cell. For example, the nucleic acid molecule encoding the modified enzyme is inserted into the expression vector and transfected into the host cell. The nucleic acid molecule encoding the modified enzyme can be transiently introduced into the host cell, or stably fused into the genome of the host cell. Standard recombinant DNA methods can be used to produce 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 exhibit 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, nucleic acids may contain protein tags, screening markers, or regulatory sequences that control protein expression in host cells, 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 well known. Those with ordinary knowledge in the technical field to which the present invention pertains can understand that the selection of expression vectors, including the selection of regulatory fragments, can be based on several factors, including the selection of the host cell to be transformed, the expression level of the required protein, etc. Exemplary regulatory sequences for mammalian host cell expression include viral fragments that direct high protein expression in mammalian cells, for example, derived from cytomegalovirus (CMV) (for example, CMV promoter and/or enhancer Simian virus 40 (Simian Virus, SV40) (for example, SV40 promoter and/or enhancer), adenovirus (for example, adenovirus major late promoter (AdMLP)) and polyoma virus The 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 can be altered, inhibited, or reduced.

c. Host cells expressing modified enzymes

Another category of the present invention relates to host cells expressing modified enzymes in the fucosylation pathway. The performance of the modified enzyme in the host cell can interfere with the activity of the wild-type enzyme, resulting in inhibition or reduction of the fucosylation pathway. Therefore, the protein (eg, antibody) produced in the host cell expressing the modified enzyme is defucosylated (afucosylated).

As used in the present invention, the phrase "hypofucosylated cell" or "hypofucosylated host cell" refers to that because the cell expresses a modified enzyme in the fucosylation pathway, fucose Cells in which the basic pathway has been inhibited or reduced.

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

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

2. Defucosylated protein

Another category of the present invention relates to a method for producing defucosylated protein. In some embodiments, the afucosylated protein is an afucosylated 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), ceruloplasmin , Α-2-macroglobulin, α-2-HS-glycoprotein, α-fetoprotein, haptoglobin, fibrinogen gamma chain precursor, immunoglobulin (including IgG, IgA, IgM, IgD, IgE And similar), APO-D, kininogen, 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 anaphylactoxin, 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 anaphylactoxin, complement C5 α'chain, complement C7, α-1 B glycoprotein, B-2-glycoprotein, vitamin D-binding Protein, Inter-α-trypsin inhibitor heavy chain H2, α-1B-glycoprotein, precursor of angiotensin, angiotensin-1, angiotensin-2, angiotensin-3, GARP protein, β -2-glycoprotein, Clusterin (Apo J), integrin alpha-8 precursor glycoprotein, integrin alpha-8 heavy chain, integrin alpha-8 light chain, hepatitis C virus particles, elf- 5. Kininogen, HSP33-homologous, lysine endopeptidase and protein 32 precursors rich in repeat 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 (e.g., multiple strains, single strains, monospecific, multispecific, chimeric, deimmunized, humanized, human, primatized, single chain, single domain, Synthetic and recombinant antibodies); part of a complete antibody with the desired activity or function (for example, an immune fragment of an antibody containing Fab, Fab', F(ab')2, Fv, scFv, single domain fragments); and Peptides and proteins of the Fc domain that can be fucosylated (for example, Fc-fusion proteins).

The term "defucosylated antibody" used in the description of the present invention refers to an antibody produced under conditions where fucosylation is inhibited or significantly reduced compared to an antibody produced under natural conditions. Fragment. The defucosylated antibody produced by the method of the present invention may be completely (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% afucosylated molecules. In other embodiments, the antibodies produced by the methods of the invention may contain about 40% to about 100% afucosylated molecules. In certain embodiments, the antibody produced by the method of the present 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% defucosylated molecules. Not all N-glycosylated antibodies or fragments thereof (for example, Fc-fusion proteins) need to be afucosylated.

b. Types of antibodies

Any antibody can be produced as an afucosylated antibody using the method of the present invention. There are no restrictions 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 antibody, anti-GD3 antibody, anti-GM2 antibody, anti-HER2 antibody, anti-CD52 antibody, anti-MAGE antibody, anti-HM124 antibody, anti-parathyroid gland Hormone-related protein (parathyroid 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 their analogs.

Examples of antibodies that recognize antigens related to 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 circulatory 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 their analogs.

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

3. Method for preparing defucosylated protein

The afucosylated protein of the present invention is produced in hypofucosylated cells, which includes afucosylated antibody. Using techniques known in the art, such as transfecting hypofucosylated cells with a protein-encoding expression vector, the defucosylated protein can be expressed in the hypofucosylated cells.

Techniques known in the art can be used to prepare protein-encoding expression vectors. For example, the amino acid sequence is reverse transcribed into a nucleic acid sequence to construct an expression vector, preferably using nucleic acid codons 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, just like the above-mentioned 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 (such as lipids, calcium phosphate, cationic polymers, DEAE-dextran, activated dendrimers, magnetic beads, etc.) can be used, and instrument-based methods (such as , Electroporation, biojet technology, microinjection, laser/photoinjection, etc.), or transfection by virus-based methods. Then, under conditions suitable for the selected expression system and host, the protein can be expressed in the transfected cells. An affinity column or other techniques known in the art can then be used to purify the expressed protein.

Transfect the nucleic acid encoding the modified enzyme (to become a hypofucosylated cell) and the nucleic acid encoding the protein (to express the protein) into the host cell in any order to produce defucosylation protein. For example, a nucleic acid encoding a modified enzyme (to be a hypofucosylated cell) can be transfected into a host cell first, and then transfected with a nucleic acid encoding a protein (the protein is to be expressed). Alternatively, the nucleic acid encoding the protein (to express the protein) can be transfected into the host cell first, and then transfected with the nucleic acid encoding the modified enzyme (to be a hypofucosylated cell). In another variation, the nucleic acid encoding the modified enzyme (to be a hypofucosylated cell) and the nucleic acid encoding the protein (to express the protein) can be transfected into the host cell at the same time.

In a specific embodiment, according to the following steps, a defucosylated protein is produced by first preparing hypofucosylated cells, and then transfecting nucleic acid encoding the protein into the hypofucosylated cells : A) Obtain a host cell suitable for expressing the protein; b) Transfect the nucleic acid encoding the modified enzyme into the host cell; c) Screen and/or isolate transfected cells with hypofucosylation; d) Add The nucleic acid encoding the protein is transfected into low-fucosylated cells; e) screening and/or isolating the low-fucosylated cells transfected with the nucleic acid encoding the protein; f) inducing the protein in low-fucosylated cells Performance in chemical cells.

In another embodiment, according to the following steps, a nucleic acid encoding a protein is first transfected into a host cell, and then the cell is transfected with a nucleic acid encoding a modified enzyme to produce afucosylated protein: a) Obtain a host cell suitable for expressing the protein; 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 a modified enzyme Transfect the nucleic acid of step (c) into the cells in step (c); e) screening and/or isolating the transfected cells with hypofucosylation in step (d); f) inducing the protein in the hypofucosylated cells In the performance.

In a variation of the above embodiment, the afucosylated protein can be produced according to the following steps: a) obtaining a host cell expressing or overexpressing the protein; b) transfecting the nucleic acid encoding the modified enzyme into the cell; c) Screening and/or isolating transfected cells with hypofucosylation; d) Inducing protein expression in hypofucosylation cells.

In another embodiment, as follows, by simultaneously transfecting the nucleic acid encoding the modified enzyme (to be a hypofucosylated cell) and the nucleic acid encoding the protein (to express the protein) to the host cell, 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) screening and/ Or isolate the transfected cells that express the protein and have hypofucosylation; d) Induce the expression of the protein in the hypofucosylation cells.

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

4. Improved properties of defucosylated antibodies

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

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

a. Increased ADCC activity

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

The "ADCC activity" used in the method of the present invention refers to the ability of an antibody to cause an antibody-dependent cellular cytotoxicity (ADCC) response. ADCC is a cell-mediated reaction in which non-specific cytotoxic cells expressing FcRs (eg, natural killer (NK) cells, neutrophils, and macrophages) recognize antibodies bound to the surface of target cells, and then create target cells Decomposition (ie, kill). The main vector cells in ADCC are natural killer (NK) cells. NK cells express FcγRIII, in which FcγRIIIA is an activating receptor and FcγRIIIB is an inhibitory receptor. Monocytes express FcyRI, FcyRII and FcyRIII. In vitro assays, such as those 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 afucosylated antibody of the present invention is at least 0.5, 1, 2, 3, 5, 10, 20, 50, 100 times that of its wild-type control antibody.

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

b. Increased CDC activity

Compared with antibodies produced using standard methods, the afucosylated antibodies of the present 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 afucosylated 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 afucosylated antibodies with enhanced CDC function. In one embodiment, the Fc variant of the present invention has increased CDC activity. In another embodiment, the afucosylated antibody has at least 2 times, or at least 3 times, or at least 5 times, or at least 10 times, or at least 50 times, or at least 100 times higher than the corresponding molecule CDC activity.

4. Use defucosylated antibodies

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

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, defucosylated antibodies are useful.

The dosage of the afucosylated antibody of the present invention will vary depending on the subject and specific mode of administration. The required dosage can be changed according to many factors well known to those with ordinary knowledge in the technical field of the present invention, including but not limited to, the antibody target, the type of the subject, and the size/weight of the subject. The dose can be 0.1 to 100,000 μg/kg body weight. The afucosylated antibody can be administered in a single dose or in multiple doses. Defucosylated antibodies can be administered once during a 24-hour period, multiple times during a 24-hour period, or continuous infusion. The afucosylated antibody 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 embodiment

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

(1) 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 defucosylation Fucosylated protein.

(2) The method of (1), wherein the modified enzyme is derived from GDP-mannose 4,6-dehydrogenase (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 fucosyltransferase (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 from which the modified enzyme is derived 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 afucosylated protein produced in the host cell is an afucosylated antibody or a fragment thereof.

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

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

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

(11) A method for producing afucosylated protein in a host cell, comprising combining 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 Transfection into host cells.

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

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

(14) The method of (11), wherein the first nucleic acid code is derived from 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 of (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) Transfect the first nucleic acid encoding the modified enzyme into host cells; b) Screen and isolate transfected cells with hypofucosylation; 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 hypofucosylated cell.

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

(18) The method according to (11), including the following steps: a) Transfect the second nucleic acid encoding the protein to be produced into the host cell; b) Screen and isolate the cells transfected with the second nucleic acid; c) Transform the encoding The first nucleic acid of the modified enzyme is transfected into the cells in step (b); d) the transfected cells with hypofucosylation are screened and isolated; e) the hypofucosylated cells are expressed to be produced的protein.

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

5. Additional Examples

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

(1) A method for producing afucosylated antibody, comprising: introducing a nucleic acid encoding at least one modified enzyme into a host cell to produce the afucosylated antibody 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 fucosyltransferase (FUT).

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

(5) The method according to (1), wherein the modified enzyme inhibits the activity of the wild-type fucosylation 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 afucosylated antibody has increased ADCC.

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

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

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

(11) 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 fucosyltransferase (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 fucosylation enzyme in the host cell.

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

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

(18) The method of (9), wherein the CDC activity of the afucosylated 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 afucosylated antibody is a human antibody or a fragment thereof.

(21) The method of (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 without or with hypofucosylation, comprising 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 enzymes in the fucosylation pathway and stable cell lines expressing the modified enzymes

1. Cell lines

Commercially available CHOdhfr(-) cell line (ATCC CRL-9096), which is a CHO cell mutant strain lacking dihydrofolate reductase activity, purchased from Culture Collection and Research Center (CCRC), Taiwan ). The CHOdhfr ^(-) cell line was divided into 3 separate cultures and processed 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 to encode HERCEPTIN® (Trastuzumab, a monoclonal antibody against the protein HER2). Obtained a stable cell line expressing HERCEPTIN® and identified it as HC59.

The third culture is untreated cells and maintained as a CHOdhfr(-) cell strain.

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

Construct many expression vectors encoding modified enzymes FUT8 and GMD.

The F83M, F8M1, F8M2, F8M3 and F8D1 mutants respectively represent different modifications to α-1,6-fucose transferase, wild-type FUT8 protein (GenBank No.NP_058589.2). 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 expressed enzymes. 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 wild-type FUT8 protein with a modified mutant type in K369E, D409K and S469V respectively. F8D1 represents the mutant type in the wild-type FUT8 protein with amino acid residues at positions 365 to 386 deleted.

Table 2 summarizes the modifications made to the wild-type nucleic acid sequence by the GMD vector and the amino acid changes produced in the expressed enzymes. In particular, mutant GMD4M represents a modification of GDP-mannose 4,6-dehydratase, wild-type GMD protein (GenBank No.NP_001233625.1, which has 4 mutations in 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 then cloned into the pHD expression vector (pcDNA3.1Hygro, Invitrogen, Carlsbad, CA, cat.no.V870-20, with dhfr Gene) Pad/EcoRv or BamHI/EcoRV position 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

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

a. RC79 cells

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

b. HC59 cells

The transfected HC59 cell line was first cultured 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 4mM Glutamax-I) EX-CELL® 325 PF CHO medium). Then, the transfected cells were cultured on EX-CELL® 325 PF containing 0.8μM MTX, 0.5mg/mL geneticin, 0.05mg/mL bleomycin, 4mM Glutamax-I and 0.25mg/mL hygromycin In CHO medium, and as described below, separated by LCA to generate a cell bank of the HC59F83M cell line.

c. CHOdhfr ^(-) cell

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

4. Isolation of low-fucosylated cells

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

All RC79, HC59 and CHO transfected strains were subjected to a preliminary selection medium containing hygromycin as selection pressure, and then used to identify the core structure of α-1,6-fucosylated trimannose, which can identify N-linked oligosaccharides. And let the cells expressing this structure go to the LCA of the cell death pathway for final screening. The transfected strains of RC79, HC59 or CHO were planted in 2.5 mL fresh medium containing 0.4 mg/mL LCA at 1.2 x 10 ^{5 cells/mL, and the cell survival rate was counted on the 3rd or 4th day.} The cells are cultured in this preliminary screening medium until the cell survival rate reaches 80%. After the cell survival rate reached 80%, the cells were resuspended in a fresh selection medium containing a gradient concentration of LCA ^{at 1.2 x 10 5 cells/mL.} The LCA screening is repeated several times until the final concentration of LCA reaches 0.6 to 1.2 mg/mL.

To analyze the amount of fucose on the cell surface, the cells were labeled with LCA and analyzed by flow cytometry. First, the cells were planted in a 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).

^{Then, wash the 1 x 10 7} cells twice with 10 mL of ice-cold PBS, and resuspend them 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).

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

5. Preparation of C109F83M cell line expressing RITUXAN®

After isolating the hypofucosylated 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 single low-fucose cell line of C109F83M, AF97, was isolated and transfected with nucleic acid encoding RITUXAN® by electroporation to express RITUXAN®. The transfected strain was transferred to a 25T flask containing non-selective medium to restore 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. The CLONEPIX TM 2 system was used to select single cells to generate AF97 anti-CD20 cell lines.

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

[Example 2] Performance and analysis of defucosylated antibodies 1. Expression and purification of antibodies

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

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

Recombinant HC59 cells were cultured in EX-CELL® 325 PF CHO medium containing 0.8μM MTX, 0.5mg/mL geneticin, 0.05mg/mL bleomycin, 4mM Glutamax-I and 0.25mg/mL hygromycin , And maintained in a shaking incubator (Infors Multitron Pro) at 37°C and 5% CO _2.

The parameters of cell culture are routinely monitored every day. Use a hemocytometer to exclude trypan blue to determine the 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. Equilibrate the protein A column with 5 column volumes of 0.1M Tris, pH 8.3, and then add the sample to the column. Wash the unbound protein with 0.1M Tris, pH 8.3 (2 times the column volume) and PBS, pH 6.5 (10 times the column volume). The column was further cleaned with 0.1M sodium acetate, pH 6.5 (10 times the column volume). Finally, the antibody was extracted with 0.1M glycine, pH 2.8, and neutralized with the same extraction volume of 0.1M Tris, pH 8.3.

2. Determination of antibody N-polysaccharide profile analysis

Analyze the N-polysaccharide profile with ACQUITY UPLC® system. First, digest 0.3 mg of antibody samples with 3 U PNGase-F in 0.3 mL digestion buffer (15 mM Tris-HCl, pH 7.0) at 37°C for 18 hours. The released N-polysaccharides were separated from the antibody by ultrafiltration using an AMICON®Ultra-0.5mL 30K device at 13,000rpm 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 (containing 0.34M aminobenzamide and 1M sodium cyanoborohydride in DMSO-acetic acid (7:3 v/v). ) Solvent) and incubate at 65°C for 3 hours. The PD MINITRAP ^TM G10 size exclusion column is used to remove excess 2-AB labeling reagent. The labeled N-polysaccharide was freeze-dried overnight and re-dissolved in 50 μL ddH ₂ O for UPLC detection. At 60°C, the N-glycan profile was obtained by ACQUITYUPLC® system and Glycan BEH Amide column. A linear gradient of 100 mM ammonium formate, pH 4.5/acetonitrile was used to separate different forms of N-glycans.

The results of flow cytometry showed that in all cell types, the binding of LCA was extremely low on the cell surface that overexpressed F83M protein. Similarly, on RC79 cells overexpressing F8M1, F8M2, F8M3, F8D1, or GMD4M protein, no LCA binding was detected (data not shown).

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

In addition, Table 4 shows RC79 cells with unmodified fucosylation pathways and RC79 cells with fucosylation pathways modified by overexpression of one of F8M1, F8M2, F8M3, F8D1 or GMD4M modified enzymes The N-glycan profile of the antibody produced in. The data in Table 4 shows that most of the anti-CD20 antibodies produced in RC79 cells with unmodified fucosylation pathway are heavily fucosylated. In particular, in these cells, only 3.67% of the anti-CD20 antibodies were afucosylated. In contrast, antibodies produced in RC79 cells that overexpress the modified enzyme have a very low degree of fucosylation. In particular, the degree of defucosylation of anti-CD20 antibodies produced by cells overexpressing F8M1, F8M2, F8M3, F8D1, or GMD4M modified enzymes ranged from 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 the FUT8 modified enzymes (F8M1, F8M2, F8M3, F8D1) is 95.70 to 97.16%, and is modified by overexpression of GMD The degree of defucosylation of the antibody produced by the enzyme (GMD4M) is 92.78%. These results confirm that the degree of deglycosylation of antibodies produced in cells overexpressing FUT8 modified enzymes is higher than that of antibodies produced in cells overexpressing 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 expressing modified enzymes (FUT8 or GMD) in the fucosylation pathway. This result also shows that the antibodies produced in these transfected cells are afucosylated antibodies.

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

The results in Table 5 show that the afucosylated antibody can be produced in cells that are first transfected with the nucleic acid encoding the modified enzyme and then transfected with the nucleic acid encoding the antibody for the second time. Therefore, the methods of the present invention can be used to modify cells to produce afucosylated antibodies.

3. The performance of FUT8 protein in recombinant cells

The precipitate of RC79 cells and recombinant cells expressing FUT8 modified enzymes (eg, F8M1, F8M2, F8M3, or F8D1) was decomposed in 1% Triton X containing a phosphatase inhibitor cocktail (Sigma-Aldrich, Cat.S8820) -100 in. The DC ^TM (cleaning agent) protein analysis (BIO-RAD) reagent was used to confirm the protein concentration in the supernatant of the decomposed cells. Each sample of the supernatant containing 30μg protein was separated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose On the 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), To block the nitrocellulose membrane for 1 hour, and separately incubate 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 with 25mM Tris-HCl (pH7.4) (containing 120mM NaCl, 0.1% gelatin(w/w) and 0.1% TWEEN® 20(v/w)) for 3 times for 5 minutes, and 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. After additional washing, SIGMAFAST DAB with Metal Enhancer (Sigma, Cat.D0426), to analyze the film.

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 of FUT8 protein in recombinant cells and RC79 parent is similar. These results indicate that the production of afucosylated antibody in RC79 cells expressing the modified enzyme has nothing to do with the expression level of FUT8 protein. These results show that FUT8 modified enzymes can interfere with the wild-type FUT8 protein in cells to inhibit and/or reduce the fucosylation pathway, so that recombinant cells can 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 the expression of the FUT8 protein, the mechanism of using the method of the present invention to produce afucosylated antibodies is novel and unique.

4. Stability of recombinant cells

To evaluate the stability of RC79 recombinant cells expressing F83M modified enzyme.

The RC79 recombinant cells were cultured in a medium without selection reagents for 3 months. Weekly flow cytometry analysis is used to monitor the fucosylation of the cells, and the ACQUITY UPLC® System and Glycan BEH Amide column are used to determine the composition of the N-glycan of the purified antibody every month, lasting 3 Month, as mentioned 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 72 days, 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 afucosylated antibodies during the 72-day study.

The 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 afucosylated antibodies for a long time.

[Example 3] ADCC activity of defucosylated antibodies

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

1. Preparation of effector cell solution

Add human peripheral blood (100 mL) from a healthy donor 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, 24mL of diluted blood is slowly added to Ficoll-Paque and centrifuged at 400 xg for 32 minutes at 25°C to separate monocytes. The buffy coat was fully distributed into two 50 mL centrifuge tubes containing 20 mL of 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 the 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 the PBMC cells. A sufficient volume of PBMC cell suspension was added to the 75T flask, and about 15 mL of cells per flask, the 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. The PBMC cells were cultured in a 37°C, 5% CO ₂ incubator for 18 hours. PBMC cells stimulated by IL-2 were collected and centrifuged at 1,200 rpm for 12 minutes at 25°C, and then the supernatant was discarded. Add PBS (10 mL) and mix with the cells. The cells were centrifuged at 1,200 rpm at 25°C for 12 minutes to remove the supernatant. The cells were resuspended in 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. The medium was analyzed with RPMI, and the cells were resuspended to prepare a target cell solution of ^{5 x 10 5 cells/mL.} Add the target cell solution (40 μL of 5 x 10 ⁵ cells/mL) to the holes of the 96-well cell culture plate at the bottom of the V. Next, add 20 μL of the prepared commercially available RITUXAN® solution (MABTHERA®) (25-0.0025μg/mL) (positive control group) and defucosylated antibody (R1 cell line) solution (25-0.0025μg). /mL), or RPMI analysis medium (negative control group) is added to the hole and mixed with the target cell solution. Incubate the V-bottom 96-well cell culture dish 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, 5% CO ₂ for 4 hours. Before collecting the supernatant, add the lysis solution (10 μL) of CYTOTOX 96® to the dish of the Tmax and BlkV groups, and react for 1 hour. Centrifuge the V-bottom 96-well cell culture dish at 300 xg for 4 minutes, and transfer 50 μL of the supernatant from the 96-well cell culture dish to the hole of the flat-bottom analysis dish.

Add lactate dehydrogenase (LDH) (2 μL) to 10 mL of LDH positive control diluent to prepare LDH positive control solution. The prepared LDH positive control solution (50 µL) was added to the holes of the 96-well flat-bottomed analysis disc.

Add the LDH reconstitution matrix mixture (50 μL) to each test hole of the analysis disc. Cover the dish and incubate in the dark at room temperature for 30 minutes. Add stop solution (50 μL) to each test hole of the plate. Immediately after adding the stop solution, the absorbance at 490 nm was recorded. Using each group (S, PBMC, T, E, and Tmax) blank removal absorbance value, ADCC activity was calculated with the following formula.

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

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

As shown in Table 7, to RC79F83MR1 fucose from cell lines of antibody-based anti -CD20 EC ₅₀ significantly lower than commercially available RITUXAN®, which is a fucosylated anti -CD20 antibody. _{In particular, the defucosylated anti-CD20 antibody (R1 cell line) has 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 ADCC activity of the defucosylated anti-CD20 antibody (R1 cell line) is 7.68 to 10.7 times that of the fucosylated anti-CD20 antibody (MABTHERA®).

[Example 4] Binding affinity of afucosylated antibody

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

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

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

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

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

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

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

[Example 5] CDC activity of defucosylated antibodies

The CDC activity of the afucosylated antibody produced by the method of the present invention was evaluated.

Daudi cells were cultured in RPMI medium, and when the cell density reached 1×10 ⁶ cells/mL, subculture was performed (subculture density: 2-3×10 ⁵ cells/mL). Daudi cells were collected and centrifuged at 300 rpm for 5 minutes. Resuspend the cells in RPMI medium to prepare ^{a cell suspension with a concentration of 1 x 10 5} cells/mL. After resuspension, 100 μL of cell suspension or 100 μL of RPMI medium was seeded into the holes of the white 96-well plate.

The commercially available RITUXAN® (MABTHERA®) and the defucosylated anti-CD20 antibody (R1 cell line) were prepared in physiological saline at a concentration of 120μg/mL to 0.234μg/mL. Next, add 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 to 96 white wells containing Daudi cells or RPMI medium In the hole of the disk. Add CELLTITER-GLO® reagent (20μL) to each hole and mix. Place the disk on a micro disk shaker, shake at 750 rpm for 2 minutes, and then incubate in the dark at room temperature for 10 minutes. Detect the luminous intensity (integration time: 1 second) by inserting a multi-mode reader with a high-sensitivity fluorescent box to calculate the EC ₅₀ value of the anti-CD20 antibody and the relevant CDC activity of the antibody.

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

GAZYVA® is a commercially available defucosylated anti-CD20 antibody, which 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 show that in order to achieve effective cancer treatment, the amount of GAZYVA® should be increased. In contrast, the results of this example and example 5 prove 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 for the efficacy of defucosylated antibodies in animal models

In this example, a subcutaneous xenograft model of B-cell lymphoma 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 expresses 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 anti-tumor efficacy of afucosylated antibody (R1 cell line) and the commercially available RITUXAN® (MABTHERA®).

The 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. The cells were resuspended in a new medium containing some conditioned medium (fresh CM: conditioned CM=9:1). The cells are subcultured at a ratio of 1:2 to 1:10 (number of cells seeded: total number of cells obtained), and the cell concentration is at least 1 x 10 ⁵ cells/mL. The culture plate was incubated at 37°C. SU-DHL-4 cells were cultured in five 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 ¹⁰⁸ cells/mL. On ice, use a pre-cooled syringe with an 18G needle to mix the cell suspension with an equal volume of MATRIGEL® in a 50 mL test tube. The final cell concentration is 5 x 10 ⁷ cells/mL. Using a pre-cooled 1mL syringe with a 23G *1" needle, inject 5 x 10 ⁷ cells/mL Matrigel-SU-DHL-4 cell mixture (100uL) subcutaneously into the back of each mouse (SCID/Beige mouse) The right side of the area. The total number of inoculated cells is 5 x 10 ⁶ cells. Use a caliper to measure the tumor volume of each mouse every 3 or 4 days, and ^{calculate with 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 200mm ³ (198.25±55.53mm ³ ), it occurred about 20 days after tumor inoculation. The mice were divided into three groups, 5 mice in each group, and then 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 every week for 3 weeks. Twice a week, the body 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, the tumor tissues were taken out, 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, using Student t test). In contrast, compared to the vehicle-only group, RITUXAN® has no statistically significant difference in tumor volume. At a dose of 1mg/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 result of tumor volume.

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

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

<110> Union Asia Pharmaceuticals United Biopharmaceuticals Peng Wenjun Chen Huirong

<120> Manufacturing method of defucosylated antibody

<130> 2018.XX.XX

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<170> PatentIn version 3.5

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Claims (16)

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, wherein the modified enzyme is derived from GDP-mannose 4,6-dehydratase (GDP-mannose 4,6-dehydratase, GMD) or fucosyltransferase (FUT) , The nucleic acid encoding the modified enzyme is selected from the group consisting of SEQ ID Nos: 3, 5, 7, 9, 11, or 15, and the modified enzyme can reduce or inhibit the modified enzyme in the host cell The activity of the wild-type enzyme from which the modified enzyme is derived. The method described in item 1 of the scope of patent application, wherein the modified enzyme is derived from fucosyltransferase (FUT). The method described in item 1 of the scope of the patent application, wherein the modified enzyme is derived from α-1,6-fucose transferase (FUT8). The method according to claim 1, wherein the modified enzyme inhibits or reduces fucosylation in the host cell. The method according to claim 1, wherein the afucosylated protein produced in the host cell is an afucosylated antibody or a fragment thereof. The method according to claim 5, wherein the defucosylated antibody or fragment thereof is at least 90% defucosylated. The method according to claim 5, wherein the defucosylated antibody has increased antibody-dependent cellular cytotoxicity (ADCC) compared to its fucosylated antibody active. The method described in item 5 of the scope of patent application, wherein the afucosylated antibody is complement dependent Complement dependent cytotoxicity (CDC), compared to its fucosylated counterpart, is not reduced and inhibited. A method for producing a defucosylated protein in a host cell, comprising combining a first nucleic acid sequence encoding a modified enzyme of a fucosylation pathway, and a second nucleic acid encoding the protein to be produced The sequence is transfected into the host cell, wherein the first nucleic acid encodes a modified enzyme derived from GDP-mannose 4,6-dehydratase (GMD) and/or fucose transferase (FUT), and the The first nucleic acid sequence is selected from the group consisting of SEQ ID Nos: 3, 5, 7, 9, 11 and/or 15. The modified enzyme can reduce or inhibit the modified enzyme in the host cell The activity of the wild-type enzyme from which it is derived. The method according to claim 9, wherein the host cell is simultaneously transfected with the first nucleic acid sequence and the second nucleic acid sequence. The method according to claim 10, wherein the afucosylated protein produced is an antibody or a fragment thereof. The method according to claim 9, wherein the first nucleic acid encodes a modified enzyme derived from α-1,6-fucose transferase (FUT8). The method described in item 9 of the scope of the patent application includes the following steps: a) transfecting the first nucleic acid encoding the modified enzyme into the host cell; b) screening and isolating hypofucosylated transfectants Transfecting the cell; c) transfecting the second nucleic acid encoding the protein to be produced into the hypofucosylated cell; d) expressing the protein to be produced in the hypofucosylated cell. The method according to item 13 of the scope of the patent application, wherein the afucosylated protein produced is an antibody and/or a fragment thereof. The method described in item 9 of the scope of patent application includes the following steps: a) transfecting the second nucleic acid encoding the protein to be produced into the host cell; b) screening and isolating the transfected second nucleic acid C) transfecting the first nucleic acid encoding the modified enzyme into the cell in step (b); d) screening and isolating transfected cells with low fucosylation; e) in the low The fucosylated cell expresses the protein to be produced. The method according to claim 15, wherein the afucosylated protein produced is an antibody and/or a fragment thereof.

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