RU2623167C2 - Fusion constructs and their application to create antibodies with high fc receptor binding affinity and with effector function - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biochemistry, in particular to the selected nucleic acid coding fusion polypeptide with beta-1.4 N-acetylglucosaminyl transferase III (GnTIII) or beta-1.4-galactosyl transferase (GalT) activity. Also the host-cell containing the specified nucleic acid and an expression vector containing the specified isolated nucleic acid are discovered. The method for glycosylation profile modification of the specified nucleic acid polypeptide is discovered.

EFFECT: invention allows for glycosylation modification in the host-cell with a view to obtaining polypeptides with enhanced therapeutic properties.

24 cl, 43 dwg, 2 tbl, ex 8

Description

The technical field of the invention

The present invention relates to the field of modification of glycosylation of proteins. More specifically, the present invention relates to nucleic acid molecules, including fusion constructs having catalytic activity, and their use for modifying glycosylation in a host cell to produce polypeptides with improved therapeutic properties, including antibodies with increased Fc receptor binding affinity and increased effector function.

State of the art

Glycoproteins mediate many essential functions in the human body, other eukaryotic organisms, and in some prokaryotes, including catalysis and signaling, intercellular interactions, recognition of molecules and their association. They make up the majority of non-cytosolic proteins in eukaryotic organisms (Lis et al, Eur. J Biochem. 218: 1-27 (1993)). Many glycoproteins have been used for therapeutic purposes, and over the past two decades, the main product of biotechnological production has been the recombinant variants of naturally occurring, secreted, glycoproteins. Examples include: erythropoietin (EP), therapeutic monoclonal antibodies (therapeutic MKAT, mAb), tissue plasminogen activator (TAII), interferon beta (IFN-β), granulocyte / macrophage colony stimulating factor (GM-CSF), human chorionic gonadotropin (HCG) (Cumming et al., Glycobiology 1: 115-130 (1991)).

The oligosaccharide component can significantly affect properties related to the effectiveness of therapeutic glycoprotein, including physical stability, resistance to proteases, interaction with the immune system, pharmacokinetics and specific biological activity. Such properties may depend not only on the presence or absence of oligosaccharides, but also on their specific structure. Parallels can be drawn between the structure of oligosaccharides and glycoprotein function. For example, certain oligosaccharide structures

by interacting with specific carbohydrate-binding proteins, the rapid elimination (clearance) of glycoprotein from the bloodstream is mediated, while others can be bound by antibodies and trigger unwanted immune responses (Jenkins et al., NatureBiotechnol 14: 975-81 (1996) )

Mammalian cells are the preferred hosts for the production of therapeutic glycoproteins due to their ability to glycosylate proteins in a manner most suitable for subsequent administration to the human body (Cumming et al., Glycobiology 1: 115-30 (1991); Jenkins et al, Nature Biotechnol. 14: 975- 81 (1996)). Bacteria extremely rarely glycosylate proteins and, like other types of conventional host cells, such as yeast, mycelial fungi, insect and plant cells, create glycosylation patterns associated with rapid clearance from the bloodstream, unwanted immune interactions and, in some specific cases, with decrease in biological activity. Over the past two decades, the most commonly used mammalian cells have been Chinese Hamster Ovary (CHO) cells. In addition to suitable glycosylation patterns, the use of these cells invariably produces genetically stable, highly productive cell clone lines. Their cultures can be brought to high densities in simple bioreactors (cultivators) using a serum-free medium, and it is possible to develop a safe reproducible biotechnological process. Other commonly used cells include hamster kidney cells (KSS), mouse NSO myeloma cells, and SP2 / 0. More recent studies relate to the production of glycoproteins using transgenic animals (Jenkins et al, NatureBiotechnol. 14: 975-81 (1996)).

All antibodies contain carbohydrate structures in the conserved positions of constant regions of the heavy chain, and each isotype (class) has a special set of N-linked (attached to the asparagine residue) carbohydrate structures, which variable affects the assembly, secretion or functional activity of the protein (Wright A., Morrison, SL, Trends Biotech. 15: 26-32 (1997)). The structure of the N-linked carbohydrate bound to the antibody can vary significantly depending on the degree of processing and may include both highly branched oligosaccharides with a high mannose content and “two-antenna” complex oligosaccharides (Wright, A., Morrison, SL, Trends Biotech. 15 : 26-32 (1997) Usually, heterogeneous processing of core (framework) oligosaccharide structures attached to a particular glycosylation site takes place, which leads to the fact that even monoclonal antibodies exist in the form of multiple glycoforms. of it is shown that there are significant differences in glycosylation among different cell lines, and slight - between one cell line cultured under different conditions (Lifely, M.R. et al, Glycobiology 5 (8):. 813-22 (1995)).

Non-conjugated monoclonal antibodies (mAbs, MKAT) may be useful as drugs for treating cancer, as evidenced by the approval by the US Food and Drug Administration (FDA) of the use of Rituximab (Rituxan ™; IDEC Pharmaceuticals, San Diego, California, and GenentechInc, San Francisco, CA) for treating CD20-positive B-cell low-grade or follicular lymphoma other than Hodgkin's lymphoma, Trastuzumab (Herceptin; GenentechInc,) for treating advanced breast cancer (Grillo-Lopez, A.- J., et al., Semis. Oncol. 26: 66-73 (1999); Goldenberg, MM, Clin. Ther. 21: 309-18 (1999 )), Gemtuzumab (Mylotarg ™, Celltech / Wyeth - Ayerst) for the treatment of recurrent acute myeloid leukemia, and Alemtuzumab (CAMPATH ™, Millenium Pharmaceuticals / Schering AG) for the treatment of B-cell chronic lymphocytic leukemia. The success of these products is the result of not only their effectiveness, but also their outstanding safety (Grillo-Lopez, A.-J., et al, Semin. Oncol. 26: 66-73 (1999); Goldenberg, MM, Clin. Ther. 21: 309-18 (1999)). Despite the achievements of these drugs, there is currently an interest in obtaining a higher specific activity of antibodies than that which can usually be achieved using therapy using unconjugated MKAT.

One way to achieve increased efficiency, while maintaining the simplicity of the production process and, if possible, avoiding significant undesirable side effects, is to enhance the natural cell-mediated effector functions of MKAT by modifying their oligosaccharide component (Umana, P. et al, Nature Biotechnol 17: 176-180 (1999)). IgG1 type antibodies most commonly used in cancer immunotherapy are glycoproteins that contain a conserved N-glycosylation site at the Asn297 position of each of the CH2 domains. The complex “two-antenna” oligosaccharides attached to Asn297 are hidden between the two CH2 domains, forming multiple contacts with the protein framework. Their presence is necessary for the implementation of the effector function of an antibody, such as antibody-dependent cellular cytotoxicity (ADCC) (Lifely, M. R., et al., Number 4 Glycobiology 5: 813-822 (1995); Jefferis, R., et al, Immunol Rev. 163: 59-76 (1998); Wright, A., Morrison, SL, Trends Biotechnol 15: 26-32 (1997)).

The authors of the present invention previously showed that increased expression of beta-1,4-N-acetylglucosaminyl transferase III (GnTIII), a glycosyl transferase that catalyzes the formation of branched (bisected) oligosaccharides, in vitro significantly increases the activity of ADCC anti-neuroblastoma chimeric monoclonal modified antibody (chCE) CHO cells (See Umana, P. et al, Nature Biotechnol. 17: 176-180 (1999), International Publication No. WO 99/54342, the full contents of each of the sources are fully incorporated into the description of the invention by reference). The chCE7 antibody belongs to an extensive class of unconjugated MKAT that possess high affinity and specificity for tumors; however, the effectiveness of these antibodies in the production of their standard industrial cell lines deficient in the GnTIII enzyme is too low for clinical use (Umana, P., et al., Nature Biotechnol. 17: 176-180 (1999)). This study showed for the first time that a strong increase in ADCC activity can be achieved by modifying antibody-producing cells for GnTIII expression, which also leads to an increase in the proportion of bisected oligosaccharides associated with the constant (Fc) region (including branched non-fucosylated oligosaccharides) compared to the level found in naturally occurring antibodies.

The results of several studies suggest that Fc receptor-dependent mechanisms contribute significantly to the action of antitumor cytotoxic antibodies, and also show that the optimal antitumor antibody should preferably bind to an activating Fc receptor and, at a minimum, to an inhibitory partner of FcγRIIB (Clynes, RA , et al., Nature Medicine 6 (4): 443-446 (2000)); Kalergis, A.M., Ravetch, J.V., J. Exp. Med. 195 (12): 1653-1659 (June 2002)). For example, the results of at least one study suggest that, in particular, the FcγRIIIa receptor has a strong influence on the effectiveness of antibody treatment (Cartron, G., et al., Blood 99 (3): 754-757 (February 2002)). This study showed that patients homozygous for the (gene) FcγRIIIa respond better to Rituximab than heterozygous patients. The authors concluded that the increased response was the result of better binding of the antibody to FcγRIIIa in vivo, which led to better ADCC activity against lymphoma cells (Cartron, G., et al., Blood 99 (3): 754-757 (February 2002) )

In addition to ADCC, successful anti-cancer antibodies often induce an Fc-independent direct signaling mechanism that determines the survival, proliferation, or death of a target cell through activation of cell signaling cascades or blocking access to growth factors (Selenko, N., et al, J Clin. Immunol. 22 (3): 124-130 (2002)). For example, it has been shown that treatment of CD20 ⁺ B cells with Rituximab induces, in addition to ADCC, complement mediated lysis and Mab-induced induction of apoptosis (Selenko, N., et al., J. Clin. Immunol. 22 (3): 124-130 (2002)). Moreover, Rituximab-induced lymphoma cell apoptosis not only kills cells, but also promotes the capture and cross-presentation of proteins - derived lymphoma cells by antigen-presenting dendritic cells, induces the maturation of dendritic cells and makes possible the emergence of specific cytotoxic T lymphocytes (CTLs) .

SUMMARY OF THE INVENTION

Given the enormous potential of antibodies with increased Fc receptor binding affinity and enhanced effector function, the present inventors have developed a method for producing such antibodies, which includes modifying the glycation profile of the Fc region of an antibody.

The present invention provides, in general terms, to provide a method for glycomodification of a host cell in order to alter the glycosylation profile of one or more polypeptides produced by this host cell. The methods of the invention can be used to produce therapeutic antibodies with altered glycosylation in the Fc region, including reduced fucosylation (transfer of fucose residues to the oligosaccharide), where, as a result of glycosylation modification, the antibodies have increased effector function and / or increased binding of the Fc receptor. The glyco-modified antibodies of the present invention are particularly useful in the therapeutic treatment of cancer in patients. In one embodiment, the host cell of the invention is subjected to glycosylation modifications by expression of a nucleic acid molecule encoding a polypeptide with catalytic activity of GnTIII or GalT. In a preferred embodiment, the fusion constructs are coexpressed together with a nucleic acid molecule encoding a polypeptide having ManII catalytic activity and / or with a nucleic acid molecule encoding a GnTII catalytic polypeptide. In another embodiment, the glyco-modified polypeptides of the present invention are produced by a host cell that has been modified to increase the expression level of a nucleic acid molecule encoding a polypeptide with ManII catalytic activity.

In another aspect, the present invention provides a host cell containing the expression vector described above containing an isolated nucleic acid containing a sequence encoding a recombinant fusion polypeptide, wherein said fusion polypeptide has beta-1,4-galactosyl transferase activity and is responsible for localization in the Golgi complex domain of any Golgi complex resident polypeptide. In one embodiment, the expression vector encodes a fusion polypeptide containing a catalytic domain of beta-1,4-galactosyl transferase III, and the domain responsible for localization in the Golgi complex is selected from the group consisting of the localization domain of mannosidase II, the localization domain of beta-1,2-N -acetylglucosaminyl transferase I, beta-1,2-N-acetylglucosaminyl transferase II localization domain, mannosidase I localization domain, and alpha 1-6 core fucosyl transferase localization domain or nucleic acid encoding a fusion polypeptide containing cat the analytical domain of beta-1,4-N-acetylglucosaminyl transferase III or beta-1,4-galactosyl transferase and responsible for localization in the Golgi complex is the domain of the heterologous Golgi resident polypeptide.

In another aspect, the present invention provides a host cell modified to express at least one nucleic acid encoding a recombinant fusion protein having beta-1,4-N-acetylglucosaminyl transferase III ("GnTIII") activity in an amount sufficient to modify oligosaccharides in an Fc polypeptide of a polypeptide produced by a host cell, wherein said polypeptide produced by said host cell is selected from the group consisting of an entire antibody molecule, an antibody fragment containing Fc region and a fusion protein containing a region equivalent to the immunoglobulin Fc region. In one embodiment, the polypeptide having GnTIII activity contains the catalytic domain of beta-1,4-N-acetylglucosaminyl transferase III and is responsible for the localization in the Golgi complex of a domain of a heterologous Golgi complex resident polypeptide selected from the group consisting of the mannosidase localization domain II, the localization domain of beta-1,2-N-acetylglucosaminyl transferase I, the localization domain of beta-1,2-N-acetylglucosaminyl transferase II, the localization domain of mannosidase I, and the alpha1-6 corefucosyl transferase localization domain.

In another aspect, the present invention provides a host cell modified to express at least one nucleic acid encoding a recombinant fusion protein having beta-1,4-galactosyltransferase ("GalT") activity in an amount sufficient to modify the oligosaccharides in Fc- a region of a polypeptide produced by a host cell, wherein said polypeptide produced by said host cell is selected from the group consisting of an entire antibody molecule, an antibody fragment containing an Fc region, and a protein fusion comprising a region equivalent to an immunoglobulin Fc-region. In one embodiment, the recombinant polypeptide having GalT activity contains a beta-1,4-galactosyltransferase catalytic domain and is responsible for localization in the Golgi complex, the domain of any heterologous Golgi complex resident polypeptide selected from the group consisting of the localization domain of mannosidase II , the localization domain of beta-1,2-N-acetylglucosaminyl transferase I, the localization domain of beta-1,2-N-acetylglucosaminyl transferase II, the localization domain of mannosidase I, and the alpha1-6 corefucosyl transferase localization domain s.

Preferably, the domain responsible for the localization in the Golgi complex is the domain of mannosidase II or beta-1,2-N-acetylglucosaminyl transferase I, or, alternatively, galactosyl transferase.

In another aspect, the present invention provides a fusion polypeptide having beta-1,4-N-acetylglucosaminyl transferase III activity and containing the domain of any heterologous Golgi complex resident polypeptide responsible for localization in the Golgi complex. In one embodiment, the fusion polypeptides of this invention comprise the catalytic domain of beta-1,4-N-acetylglucosaminyl transferase III. In another embodiment, the localization domain in the Golgi complex is selected from the group consisting of the localization domain of mannosidase II, the localization domain of beta-1,2-N-acetylglucosaminyl transferase I, the localization domain of mannosidase I, the localization domain of beta-1,2-N-acetylglucosaminyl transferase II and the localization domain of alpha 1-6 core-fucosyltransferase.

In another aspect, the present invention provides a fusion polypeptide having beta-1,4-galactosaltransferase activity and containing a domain responsible for the localization in the Golgi complex of any heterologous Golgi resident polypeptide. In one embodiment, the fusion polypeptides of the invention comprise a beta-1,4 galactosyl transferase catalytic domain. In another embodiment, the domain responsible for localization in the Golgi complex is selected from the group consisting of the localization domain of mannosidase II, the localization domain of beta-1,2-N-acetylglucosaminyl transferase I, the localization domain

mannosidase I, the localization domain of beta-1,2-N-acetylglucosaminyl transferase II and the localization domain of alpha 1-6 core-fucosyl transferase.

Preferably, the localization site in the Golgi complex is the domain of mannosidase II or beta-1,2-N-acetylglucosaminyl transferase I ("GnTI"), or, alternatively, galactosyl transferase ("GalT").

In another aspect, the present invention provides a method for producing a recombinant fusion polypeptide having beta-1,4-N-acetylglucosaminyl transferase III activity, comprising culturing a host cell of the invention in a culture medium under conditions that allow expression of a nucleic acid encoding the fusion polypeptide, and obtaining (isolating) the polypeptide from the resulting cell culture. In one embodiment, the fusion polypeptide comprises a catalytic domain of beta-1,4-N-acetylglucosaminyl transferase III. Preferably, the fusion polypeptide contains the localization in the Golgi complex domain of a heterologous resident protein of the Golgi complex, which is selected from the group consisting of the localization domain of mannosidase I, the localization domain of beta-1,2-N-acetylglucosaminyl transferase I, the localization domain of mannosidase I, the localization domain of beta-1,2-N-acetylglucosaminyl transferase II and the localization domain of alpha1-6 core-fucosyl transferase.

In another aspect, the present invention provides a method for producing a recombinant fusion polypeptide having beta-1,4-N-galactosyl transferase activity, comprising culturing in a host cell medium according to the present invention under conditions that allow expression of a nucleic acid encoding a fusion polypeptide and obtaining ( isolation) a fusion polypeptide from the resulting culture. In one embodiment, the fusion polypeptide comprises a beta-1,4-N-galactosyl transferase catalytic domain. Preferably, the fusion polypeptide contains the localization domain in the Golgi complex of a domain of a heterologous resident protein of the Golgi complex, which is selected from the group consisting of the localization domain of mannosidase II, the localization domain of beta-1,2-N-acetylglucosaminyl transferase I, the localization domain of mannosidase The domain of localization of beta-1,2-N-acetylglucosaminyltransferase II and the localization domain of alpha1-6 corefucosyltransferase.

Preferably, the localization domain in the Golgi complex is the domain of mannosidase II, or beta-1,2-N-acetylglucosaminyl transferase I ("GnTI"), or galactosyl transferase ("GalT").

In a further aspect, the invention provides a method for modifying the glycosylation profile of a polypeptide produced by a host cell containing at least one nucleic acid or expression vector of the invention introduced into it. Preferably, the polypeptide is IgG or a fragment thereof containing the Fc region of this polypeptide (IgG). In a particularly preferred embodiment, the polypeptide is IgG or a fragment thereof comprising the Fc region of the polypeptide. Alternatively, the polypeptide is a fusion protein that includes a region equivalent to the Fc region of human IgG.

In another aspect, the present invention provides a method for producing a recombinant fusion polypeptide in a host cell, comprising: (a) culturing a host cell modified to express at least one nucleic acid encoding a fusion polypeptide having beta-1,4-N- activity acetylglucosaminyl transferase III ("GnTIII") or, alternatively, beta-1,4-galactosyl transferase ("GalT") activity in a culture medium under conditions that allow the production of a polypeptide selected from the group consisting of of whole antibody molecule, an antibody fragment containing the Fc-fragment and the fusion protein comprising a region equivalent to an immunoglobulin Fc-region, wherein said fusion polypeptide is expressed in an amount sufficient to modify the oligosaccharides in the Fc-region of a polypeptide produced by the host cell; and (b) isolating said polypeptide. Preferably, the fusion polypeptide contains a catalytic domain of beta-1,4-N-acetylglucosaminyl transferase III or beta-1,4-galactosyl transferase ("GalT") and further comprises a domain in a Golgi complex for a heterologous resident polypeptide of the Golgi complex which is selected from the group consisting of the localization domain of mannosidase II, the localization domain of beta-1,2-N-acetylglucosaminyl transferase I, the localization domain of mannosidase I, the localization domain of beta-1,2-N-acetylglucosaminyl transferase II and the loc domain alization of alpha 1-6 bovine fucosyl transferase. In preferred embodiments, the polypeptide produced by the host cell has, as a result of the modification, increased effector function and / or increased binding of the Fc receptor. In particularly preferred embodiments, increased effector function is increased Fc-mediated cell cytotoxicity, increased NK cell binding, increased macrophage binding, increased monocyte binding, increased polymorphonuclear cell binding, increased direct transmission of apoptosis-inducing signals, increased dendritic cell maturation and / or increased dendritic cell maturation activity of the initial activation of T-lymphocytes (priming), and increased binding of the Fc receptor is an increased binding of the activating Fc-receptor, such as FcγRIIIA receptor. Preferably, a polypeptide exhibiting increased effector function and / or increased binding of the Fc receptor is an antibody, antibody fragment or fusion protein containing a region equivalent to the immunoglobulin Fc region and has an increased proportion of unfucosylated oligosaccharides in the Fc region.

In a further aspect, the invention provides pharmaceutical compositions comprising an antibody, an antibody fragment containing an Fc region, or a recombinant fusion polypeptide containing the immunoglobulin Fc region of the invention, and the use of such compositions in the treatment of tumors such as cancer or other disorders. In one embodiment, the treatment is a reduction in the number of B lymphocytes by administering a therapeutically effective amount of such a pharmaceutical composition to a patient, for example, a human, if necessary. In a further aspect, the invention provides a host cell containing an expression vector containing a nucleic acid molecule encoding a fusion polypeptide, wherein said fusion polypeptide has beta-1,4-N-acetylglucosaminyl transferase III (GnTIII) activity and contains a domain responsible for localization in the Golgi complex any Golgi complex resident polypeptide, as well as an expression vector containing a nucleic acid molecule encoding a polypeptide, wherein said fusion polypeptide has manno activity idazy II. In preferred embodiments, the fusion polypeptide contains a GnTIII catalytic domain, and the Golgi complex localization domain is selected from the group consisting of the ManII localization domain, the GnTI localization domain, the GnTII localization domain, the ManI localization domain, and the alpha-1-6 corefucosyl transferase localization domain. In one embodiment, the host cell further comprises an expression vector encoding a polypeptide having GnTII activity. Nucleic acid molecules encoding a fusion polypeptide having a ManII polypeptide activity and having a GnTII polypeptide activity can be contained either in different expression vectors or in one expression vector. In an additional aspect, the invention provides a host cell containing an expression vector containing a nucleic acid molecule encoding a recombinant fusion polypeptide, wherein said fusion polypeptide has beta-1,4-galactosyl transferase (GalT) activity and contains the polypeptide domain responsible for the localization of the Golgi complex a resident of the Golgi complex, as well as an expression vector containing a nucleic acid molecule encoding a polypeptide, wherein said polypeptide has mannosidase II (ManII) activity. In preferred embodiments, the fusion polypeptide contains a GnTIII catalytic domain, and the Golgi complex localization domain is selected from the group consisting of the ManII localization domain, the GnT I localization domain, the GnTII localization domain, the ManI localization domain, and the CORI fucosyltransferase alpha1-6 localization domain. In one embodiment, the host cell further comprises an expression vector encoding a polypeptide having GnTII activity. Nucleic acid molecules encoding a fusion polypeptide having ManII polypeptide activity and GnTII polypeptide activity can be contained either in different expression vectors or in one expression vector.

In a further aspect, the invention provides a host cell modified to express at least one nucleic acid encoding a fusion polypeptide having GnT III activity and at least one nucleic acid encoding a polypeptide having Man II activity in an amount sufficient to modify the oligosaccharides in the Fc region of a polypeptide produced by a host cell, wherein said polypeptide produced by said host cell is selected from the group consisting of a whole antibody molecule, fr an antibody component containing an Fc fragment and a fusion protein that contains a region equivalent to the immunoglobulin Fc region.

In an additional embodiment, the invention provides a host cell modified to express at least one nucleic acid encoding a fusion polypeptide having GnT III activity, at least one nucleic acid encoding a polypeptide having Man II activity, and at least one nucleic acid an acid encoding a polypeptide having GnT II activity in an amount sufficient to modify the oligosaccharides in the Fc region of the polypeptide produced by the specified host cell, where nny polypeptide produced by said host cell is selected from the group consisting of a whole antibody molecule, an antibody fragment, and a fusion protein that includes a region equivalent to an immunoglobulin Fc-region.

In a further aspect, the invention provides a host cell modified to express at least one nucleic acid encoding a fusion polypeptide having GalT activity, and at least one nucleic acid encoding a polypeptide having Man II activity, in an amount sufficient to modify the oligosaccharides in the Fc region of a polypeptide produced by a host cell, wherein said polypeptide produced by said host cell is selected from the group consisting of an entire antibody molecule, fra an antibody fragment and a fusion protein that contains a region equivalent to the immunoglobulin Fc region. In an additional aspect, the invention provides a host cell modified to express at least one nucleic acid encoding a recombinant fusion polypeptide having GalT activity, at least one nucleic acid encoding a polypeptide having Man II activity, and at least one nucleic acid encoding a polypeptide having GnT II activity in an amount sufficient to modify the oligosaccharides in the Fc region of the polypeptide produced by the specified host cell, where The indicated polypeptide produced by said host cell is selected from the group consisting of a whole antibody molecule, an antibody fragment, and a fusion protein that contains a region equivalent to the immunoglobulin Fc region.

In a further aspect, the invention provides a method for producing a polypeptide by a host cell, comprising culturing a host cell modified to express at least one nucleic acid encoding a fusion polypeptide having GnT III activity and at least one nucleic acid encoding a polypeptide having activity Man II, under conditions that allow the production of a polypeptide selected from the group consisting of a whole antibody molecule, an antibody fragment and a fusion protein that win region equivalent to an immunoglobulin Fc-region, wherein said fusion polypeptide is expressed in an amount sufficient to modify the oligosaccharides in the Fc-region of said polypeptide produced by the host cell and recovering said polypeptide.

In another aspect, the invention provides a method for producing a polypeptide by a host cell, comprising culturing a host cell modified to express at least one nucleic acid encoding a fusion polypeptide having GalT activity, and at least one nucleic acid encoding a polypeptide having Man activity II, under conditions that allow the production of a polypeptide selected from the group consisting of a whole antibody molecule, an antibody fragment and a fusion protein that contains it is a region equivalent to the immunoglobulin Fc region, wherein said fusion polypeptide is expressed in an amount sufficient to modify oligosaccharides in the Fc region of said polypeptide produced by said host cell, as well as isolation of said polypeptide.

In an additional aspect, the invention provides a method for producing a polypeptide having increased Fc-mediated cell cytotoxicity, a host cell, comprising culturing a host cell modified to express at least one nucleic acid encoding GalT, and at least one nucleic acid encoding Man II, under conditions that allow the production of a polypeptide selected from the group consisting of a whole antibody molecule, an antibody fragment that contains the Fc region of imm noglobulina, wherein the level of expression of one of GalT and Man II enzymes, or both is sufficient to modify the oligosaccharides in the Fc-region of said polypeptide produced by said host cell and wherein said polypeptide possesses, as a result of said modification, increased Fc-mediated cellular cytotoxicity.

In another aspect, the present invention provides a method for producing a polypeptide by a host cell, comprising: (a) culturing a host cell modified to express at least one nucleic acid encoding a polypeptide having alpha-mannosidase I activity under conditions that allow the production of the polypeptide selected from the group consisting of an entire antibody molecule, an antibody fragment, and a fusion protein that contains a region equivalent to the immunoglobulin Fc region, wherein said polyp ptid having the activity of alpha-mannosidase II, expressed in an amount sufficient to modify the oligosaccharides in the Fc-region of said polypeptide produced by said host cell; and (b) isolating said polypeptide produced by said host cell.

In another aspect, the invention provides a host cell modified to express at least one nucleic acid encoding a polypeptide having alpha-mannosidase II activity under conditions that allow for the production of a polypeptide selected from the group consisting of an entire antibody molecule, an antibody fragment, and a protein a fusion that contains a fragment equivalent to an immunoglobulin Fc fragment, wherein said polypeptide having alpha-mannosidase II activity is expressed in an amount sufficient to modify the oligosaccharides in the Fc-fragment of the polypeptide produced by the host cell.

In yet another aspect, the present invention provides polypeptides produced by such a host cell, in particular antibodies having, as a result of said modification of oligosaccharides, enhanced effector function and / or increased Fc receptor binding.

Brief Description of Drawings

FIG. 1: Spectrum of a mixture of neutral oligosaccharides of a recombinant unchanged (non-glyco-modified) anti-CD20 IgG1 class antibody produced by BHK obtained on a MALDI / TOF-MS mass spectrometer. Cells were transfected with the antibody expression vector pETR1502. Purification of the antibody from the culture medium, as well as the preparation and analysis of oligosaccharides was carried out as described in the Materials and Methods section of Example 1.

FIG. 2: MALDI / TOF-MS mass spectrometer spectrum of a mixture of neutral oligosaccharides of a recombinant glyco-modified anti-CD20 IgG1 antibody produced by BHK cells modified with a nucleic acid encoding wild-type GnTIII ("wt"). Cells were co-transfected with the antibody expression vector pETR1502 and the GnTIII expression vector pETR1166. Purification of the antibody from the culture medium, as well as the preparation and analysis of oligosaccharides was carried out as described in the Materials and Methods section of Example 1.

FIG. 3: Spectrum of a mixture of neutral oligosaccharides of a recombinant glyco-modified anti-CD20 IgG1 antibody produced by BHK cells modified with a nucleic acid encoding a fusion polypeptide (Gl-GnTIII) with functional GnTIII activity obtained on a MALDI / TOF-MS mass spectrometer for localization in the Golgi complex, the GnTI domain. Cells were co-transfected with the antibody expression vector pETR1502 and the GnTIII expression vector pETR1425. Purification of the antibody from the culture medium, as well as the preparation and analysis of oligosaccharides was carried out as described in the Materials and Methods section of Example 1.

FIG. 4: Spectrum of a mixture of neutral oligosaccharides of a recombinant glyco-modified anti-CD20 IgG1 antibody produced by BHK cells modified with a nucleic acid encoding a fusion polypeptide ("M2-GnTIII") with GnTIII activity obtained on a MALDI / TOF-MS mass spectrometer localization in the Golgi complex of the domain of mannosidase I. Cells were co-transfected with the antibody expression vector pETR1502 and the GnTIII expression vector pETR1506. Purification of the antibody from the culture medium, as well as the preparation and analysis of oligosaccharides was carried out as described in the Materials and Methods section of Example 1.

FIG. 5: Spectrum of a mixture of neutral oligosaccharides of a recombinant unchanged (non-glyco-modified) anti-CD20 IgG1 antibody produced by HEK293-EBNA cells obtained on a MALDI / TOF-MS mass spectrometer. Cells were transfected with the antibody expression vector pETR1502. Purification of the antibody of the culture medium, as well as the preparation and analysis of oligosaccharides was carried out as described in the Materials and Methods section of Example 1.

FIG. Figure 6: MALDI / TOF-MS mass spectrometer spectrum of a mixture of neutral oligosaccharides of a recombinant glyco-modified anti-CD20 IgG1 antibody produced by HEK293-EBNA cells modified with a nucleic acid encoding a fusion polypeptide ("M2-GnTIII") with GnT activity functional alpha-mannosidase II domain responsible for localization in the Golgi complex. Cells were co-transfected with the antibody expression vector pETR1520 and the GnTIII expression vector pETR1519. Purification of the antibody from the culture medium, as well as the preparation and analysis of oligosaccharides was carried out as described in the Materials and Methods section of Example 1.

FIG. 7: Spectra obtained on a MALDI / TOF-MS mass spectrometer of a mixture of neutral oligosaccharides of a recombinant glyco-modified anti-CD20 IgG1 antibody produced by HEK293-EBNA cells modified with a nucleic acid encoding a fusion polypeptide ("M2-GnTIII") with GnTIII activity functional alpha-mannosidase II domain responsible for localization in the Golgi complex. Cells were co-transfected with the antibody expression vector pETR1520 and the GnTIII expression vector pETR1519. Purification of the antibody from the culture medium, as well as the preparation and analysis of oligosaccharides was carried out as described in the Materials and Methods section of Example 1. (a) The composition of oligosaccharides formed upon treatment with PNGaseF glycosidase without further enzymatic treatment. (b) Composition of oligosaccharides formed upon treatment with PNGaseF glycosidase after further digestion with EndoH endoglycosidase.

FIG. 8: (a) Schematic description of endoglycosidase-catalyzed EndoH oligosaccharide cleavage. Endoglycosidase EndoH is capable of cleaving hybrid (and branched hybrid), but not complex or complex branched oligosaccharides. (b) Due to the fact that EndoH endoglycosidase is able to “distinguish” complex and hybrid oligosaccharides, EndoH treatment allows one to determine which structures correspond to peaks in the spectrum of oligosaccharides with the same m / z ratios in the MALDI / TOF-MS spectra originally obtained as a result of processing with endoglycosidase PNGase

FIG. 9: Antibody-dependent cell cytotoxicity (ADCC), glyco-modified with the “G1-GnTIII” recombinant anti-CD20 chimeric IgG1 antibody compared to the ADCC unchanged recombinant anti-CD20 chimeric IgG1 antibody. Both antibodies were produced by BHK cells. The preparation and glycosylation profile of a glyco-modified antibody is depicted in FIG. 3, and the preparation and glycosylation profile of an unchanged antibody is shown in FIG. 1. Target cells (T) were SKW6.4 human lymphoblastoid cells. Effector cells (E) were freshly isolated human PBMCs. Cells E and T were used (with respect to E: T = 25: 1) in an ADCC assay with a 4-hour incubation that determined cytotoxicity in relation to the release of lactate dehydrogenase (LDH) to the controls for maximum release (when using detergent instead of antibody) and spontaneous release ( instead of antibody, culture medium). Details of the analysis are described in the Materials and Methods section of Example 1.

FIG. 10: Antibody-dependent cell cytotoxicity (ADCC) of glycogen-modified using the “M2-GnTIII” recombinant anti-CD20 chimeric IgG1 antibody compared to the ADCC of an unchanged recombinant anti-CD20 chimeric IgG1 antibody. Both antibodies were produced by HEK293-ENBA cells. The preparation and glycosylation profile of a glyco-modified antibody is depicted in FIG. 6, and the unchanged antibody in FIG. 5. The target cells (T) were SKW6.4 human lymphoblastoid cells. Effector cells (E) were freshly isolated human PBMCs. Cells E and T were used (with respect to E: T = 25: 1) in an ADCC assay with a 4-hour incubation that determined ADCC for the ratio of lactate dehydrogenase (LDH) release to maximal release controls (when using detergent instead of antibody) and spontaneous release ( instead of antibody, culture medium). Details of the analysis are described in the Materials and Methods of Example 1 section.

FIG. 11: Antibody-dependent cell cytotoxicity (ADCC) of glyco-modified with the "M2-GnTIII" recombinant anti-CD20 chimeric IgG1 antibodies compared to ADCC with the glyco-modified with "wt-GnTIII" recombinant anti-CD20 chimeric IgG1 chimeric. Both antibodies were produced by HEK293-ENBA cells. The preparation and glycosylation profile of a glyco-modified antibody is depicted in FIG. 4, and glyco-modified with the wt-GnTIII antibody, in FIG. 2. Target cells (T) were SKW6.4 human lymphoblastoid cells. Effector cells (E) were freshly isolated human PBMCs. Cells E and T were used (with respect to E: T = 25: 1) in an ADCC assay with a 4-hour incubation that determined ADCC for the ratio of lactate dehydrogenase (LDH) release to maximal release controls (when using detergent instead of antibody) and spontaneous release ( instead of antibody, culture medium). Details of the analysis are described in the Materials and Methods section of Example 1.

FIG. 12: Binding of the FcgammaRIIIa receptor on NK cells with a glyco-modified “M2-GnTIII” recombinant IgG1 chimeric anti-CD20 antibody, compared to the same receptor binding of an unchanged recombinant IgG1 chimeric anti-CD20 antibody. Both antibodies were produced by HEK293-ENBA cells. The preparation and glycosylation profile of a glyco-modified antibody is depicted in FIG. 6, and the unchanged antibody in FIG. 5. The binding analysis was carried out as described in the Materials and Methods section of Example 1. Human NK cells expressing the FcgammaRIIIa receptor on their surface were isolated from the donor organism with a genotype excluding the production of the FcgammaRIIc receptor (that is, a homozygous variant of the gene which contains the intramrame stop codon within the sequence encoding FcgammaRIIc). The geometric mean fluorescence intensity, measured by the method of sorting cells with activation of fluorescence (FACS) using fragments of anti-human IgG antibodies labeled with FITC (fluorescein isothiocyanate), will increase with an increase in the number of NK-linked recombinant antibodies. As shown by the use of a competing fragment of an FcgammaRIIa-specific antibody, the binding determined in this assay is FcgammaRIIIa-specific (See FIG. 13).

FIG. 13: Binding of the FcgammaRIIIa receptor on NK cells by a glyco-modified “M2-GnTIII” recombinant IgG1 chimeric anti-CD20 antibody compared to binding of the same receptor by unchanged recombinant chimeric anti-CD20 IgG1 class antibody in the presence of increasing concentrations of competing IgG1 specific fragment II antibodies. Both recombinant antibodies were produced by HEK293-ENBA cells. The preparation and glycosylation profile of a glyco-modified antibody is depicted in FIG. 6, and the unchanged antibody in FIG. 5. The binding assay was performed as described in the Materials and Methods section of Example 1, but incubating the purified NK cells simultaneously with the recombinant antibody (always at a final concentration of 3 μg / ml) and with a fragment of the competing anti-FcgammaRIII antibody 3G8-Fab2 in increasing and changing concentrations (see graph). Human NK cells expressing the FcgammaRIIIa receptor on their surface were isolated from a donor organism with a genotype excluding the production of the FcgammaRIIc receptor (i.e., a homozygous variant variant gene that contains an intra-frame stop codon within the sequence encoding FcgammaRIIc). The geometric mean fluorescence intensity, measured by the method of sorting cells with activation of fluorescence (FACS) using fragments of anti-human IgG antibodies labeled with FITC (fluorescein isothiocyanate), will increase with an increase in the number of NK-cells bound recombinant antibodies.

FIG. 14: Spectra of mixtures of neutral oligosaccharides of recombinant IgG1 “L19” class antibodies recognizing the ED-B + fibronectin isoform and produced by HEK293-ENBA cells obtained on a MALDI / TOF-MS mass spectrometer. (a) The unchanged antibody produced by HEK293-ENBA cells transfected with the antibody expression vector pETR1546. (b) An antibody glyco-modified with M2-GnTIII produced by HEK293-ENBA cells cotransfected with the antibody expression vector pETR1546 and the GnTIII expression vector pETR1519. Both antibodies were purified from the culture medium by protein A affinity chromatography followed by size exclusion chromatography on a Superdex200 matrix (Amersham), when the buffer was replaced with an aphosphate buffer solution (PBS). The preparation and analysis of oligosaccharides was carried out as described in the section "Materials and methods" of Example 1.

FIG. 15: Binding of the FcgammaRIIb receptor on Raji lymphoid cells (Human Burkitt's Lymphoma) with glyco-modified “M2-GnTIII” recombinant anti-ED-B + -fibronectin IgG1 antibody in comparison with binding of the same receptor to unchanged recombinant anti-ED-B + fibronectin with an IgG1 class antibody. Both antibodies were produced by HEK293-ENBA cells. The preparation and glycosylation profile of a glyco-modified antibody is depicted in FIG. 14b, and the unmodified antibody in FIG. 14a. Binding analysis was performed as described in the “Materials and Methods” section of Example 1. The geometric mean fluorescence intensity measured by “fluorescence activated cell sorting” (FACS) using fragments of anti-human IgG antibody labeled with FITC (fluorescein isothiocyanate) will increase with an increase in the amount of Raji line B-cell-associated lymphoma.

FIG. 16: Binding of the FcgammaRIIIa receptor on NK cells of different donors by a glyco-modified “M2-GnTIII” recombinant IgG1 anti-CD20 antibody compared to binding of the same receptor by an unchanged IgG1 recombinant anti-CD20 antibody. Both recombinant antibodies were produced by HEK293-ENBA cells. The preparation and glycosylation profile of a glyco-modified antibody is depicted in FIG. 6, and the unchanged antibody in FIG. 5. The binding analysis was performed as described in the Materials and Methods section of Example 1. Human NK cells expressing the FcgammaRIIIa receptor on their surface were isolated from the donor organism with a genotype excluding the production of the FcgammaRIIc receptor (that is, a homozygous variant of the gene which contains the intramrame stop codon within the sequence encoding FcgammaRIIc). Genotyping of two donors showed that they are homozygous for the “high affinity” variant of the FcgammaRIIIa gene (158V). Genotyping of the other two donors showed that they are heterozygous (ie their genotype is 158V / F, where 158V stands for “high affinity” and 158F stands for “low affinity” variants of the FcgammaRIIIa receptor). The geometric mean fluorescence intensity, measured by the method of sorting cells with activation of fluorescence (FACS) using a fragment of anti-human IgG antibodies labeled with FITC (fluorescein isothiocyanate), will increase with increasing number associated with NK cells of the recombinant antibody. As shown by the use of a competing fragment of an FcgammaRIIIa-specific antibody, the binding determined in this assay is FcgammaRIIIa-specific (See FIG. 13).

FIG. 17: FACS analysis of the expression of truncated CD4 (tCD4) (a) cells BHK-1502-28 (wild type) and (b) clone VKH-1502-28-11 (glycogen modified "M2-GnTIII") - a stable cell line producing chimeric anti-CD20 IgG1 class antibody. The expression of tCD4 is functionally related to the expression of M2-GnTIII through the IRES element of the GnTIII expression vector pETR1537, and thus can be used as an indirect marker of GnTIII expression. The mean and geometric mean fluorescence intensities were 27.6 and 19.9, respectively, for the glyco-modified cell line and 4.7 and 4.1 for the wild-type cell line.

FIG. 18: MALDI / TOF-MS mass spectrometer spectrum of a mixture of neutral oligosaccharides glycomodified with M2-GnTII recombinant anti-CD20 chimeric IgG1 antibody produced by VPK-1502-28-11 cell line. The cell line, antibody purification, and the preparation and analysis of oligosaccharides are described in the Materials and Methods section of Example 1.

FIG. 19: Spectrum of a mixture of neutral oligosaccharides obtained on a MALDI / TOF-MS mass spectrometer obtained by releasing oligosaccharides by treatment with PNGase glycosidase followed by treatment with EndoH endoglycosidase from a glycol-modified M2-GnTIII recombinant anti-CD20 antibody class VNK-1502-28-11. The cell line, antibody purification, and the preparation and analysis of oligosaccharides are described in the Materials and Methods section of Example 1.

FIG. 20: Binding of the FcgammaRIIIa receptor on NK cells by glycogen modified with the "M2-GnTIII" recombinant IgG1 chimeric anti-CD20 antibody compared to the same receptor binding of an unchanged recombinant IgG1 chimeric anti-CD20 antibody. (antibodies are produced by stable cell lines). The glycosylation profile of the glyco-modified antibody is shown in FIG. 18 and FIG. 19. The binding analysis was carried out as described in the Materials and Methods section of Example 1. Human NK cells expressing the FcgammaRIIa receptor on their surface were isolated from the donor organism with a genotype excluding the production of the FcgammaRIIc receptor (ie, a homozygous variant of the gene which contains the intramrame stop codon within the sequence encoding FcgammaRIIc). The geometric mean fluorescence intensity, measured by the method of sorting cells with activation of fluorescence (FACS) using a fragment of anti-human IgG antibodies labeled with FITC (fluorescein isothiocyanate), will increase with increasing number of NK-associated recombinant antibodies. As shown by the use of a competing fragment of an FcgammaRIIa-specific antibody, the binding determined in this assay is FcgammaRIIIa-specific (See FIG. 13).

FIG. 21. Complement-mediated lysis (here KOL) of glycodiagnosed using “M2-GnTIII" recombinant chimeric anti-CD20 IgG1 antibody in comparison with KOL unchanged recombinant chimeric anti-CD20 IgG1 antibody. Both antibodies were produced by HEK293-ENBA cells. The preparation and glycosylation profile of a glyco-modified antibody is depicted in FIG. 6, and the unchanged antibody in FIG. 5. The target cells (T) were SKW6.4 human lymphoblastoid cells. A human complement was used in the analysis. Lysis was measured by the release of LDH. Details of the analysis are described in the Materials and Methods section of Example 1.

FIG. 22: Spectra of mixtures of neutral oligosaccharides of recombinant chimeric IgG1 “C225” class antibodies obtained by the epidermal growth factor receptor (REFR) and produced by HEK293-ENBA cells obtained on a MALDI / TOF-MS mass spectrometer. (a) An unmodified antibody produced by HEK293-ENBA cells transfected with the pETRURSI28 antibody expression vector (b) a glyco-modified M2-GnTIII antibody produced by HEK293-ENBA cells co-transfected with the pETRURSI19T8 antibody expression expression vector pETRURSI28 and vector. Both antibodies were purified from the culture medium by protein A affinity chromatography followed by size exclusion chromatography on a Superdex200 matrix (Amersham), replacing the buffer with phosphate buffered saline (PBS). The preparation and analysis of oligosaccharides was carried out as described in the section "Materials and methods" of Example 1.

FIG. 23: Antibody-dependent cell cytotoxicity (ADCC) of a glycodiagnosed with M2-GnTIII recombinant anti-REFR chimeric IgG1 antibody “C225” compared to the ADCC of an unchanged recombinant anti-REFR chimeric IgG1 “C225” antibody. Both antibodies were produced by HEK293-ENBA cells. The preparation and glycosylation profile of a glyco-modified antibody is depicted in FIG. 22a, and the unchanged antibody in FIG. 22b. Target cells (T) were A431 human squamous cell carcinoma cells (ECACC No. 85090402). Effector cells (E) were freshly isolated human PBMCs. Cells E and T were used (with respect to E: T = 25: 1) in an ADCC assay with a 4-hour incubation that determined ADCC for the ratio of lactate dehydrogenase (LDH) release to maximal release controls (when using detergent instead of antibody) and spontaneous release ( instead of antibody, culture medium). Details of the analysis are described in the Materials and Methods section of Example 1.

FIG. 24: Nucleic acid sequence and amino acid sequence, respectively, of the ManII-GnTIII fusion polypeptide of the present invention.

FIG. 25: Nucleic acid sequence and amino acid sequence, respectively, of the GnTI-GnTIII fusion polypeptide of the present invention.

FIG. Figure 26: MALDI / TOF-MS mass spectrometer spectrum of a mixture of neutral oligosaccharides of unchanged recombinant anti-CD20 chimeric IgG1 class C2C8 antibody ("Cwt") produced by HEK293-EBNA cells transfected with the antibody expression vector pETR1520. Purification of antibodies from the culture medium, as well as the preparation and analysis of oligosaccharides are described in the section "Materials and methods" of Example 5.

FIG. Figure 27: MALDI / TOF-MS mass spectrometer spectrum of a mixture of neutral oligosaccharides of a glyco-modified recombinant anti-CD20 chimeric IgG1 class C2C8 antibody ("Cbrt") produced by HEK293-EBNA cells co-transfected with the expression vector of the PETR1520 polypeptide of expression of the G vector and polypeptide GET polypeptide 15 Gt pETR1519. Purification of the antibody from the culture medium, as well as preparation and analysis of oligosaccharides was carried out as described in the Materials and Methods section of Example 5. (A) Composition of oligosaccharides released upon PNGaseF glycosidase treatment without further enzymatic treatment. (B) Composition of oligosaccharides released upon treatment with PNGaseF glycosidase after further cleavage with EndoH endoglycosidase.

FIG. Figure 28: MALDI / TOF-MS mass spectrometer spectrum of a mixture of neutral oligosaccharides of a glyco-modified recombinant anti-CD20 chimeric IgG1 class C2C8 antibody ("Cm") produced by HEK293-EBNA cells co-transfected with the expression vector of the PETR1520 polypeptide of expression of the vector polypeptide GET polytene III vector (pETR1519) and an expression vector for the mannosidase II polypeptide (pCLF9). Purification of the antibody from the culture medium, as well as preparation and analysis of oligosaccharides was carried out as described in the Materials and Methods section of Example 5. (A) Composition of oligosaccharides released upon PNGaseF glycosidase treatment without further enzymatic treatment. (B) Composition of oligosaccharides released upon treatment with PNGaseF glycosidase after further cleavage with EndoH endoglycosidase.

FIG. 29: Antibody-dependent cell cytotoxicity (ADCC) mediated by chimeric anti-CD20 antibodies glyco-modified by expression in HEK293-EBNA cells of a nucleic acid encoding the ManII-GnTIII fusion protein, where the GnTIII catalytic domain is localized in the ManII complex, where the Golgi complex is located The -GnTIII nucleic acid is expressed either alone (Cbrt) or together with a nucleic acid encoding ManII (Cm). Cwt is an unchanged recombinant anti-CD20 chimeric IgG1 class C2C8 antibody ("Cwt") produced by HEK293-EBNA cells transfected with the antibody expression vector pETR1520. Details of the analysis are described in the Materials and Methods section of Example 1.

FIG. 30: Binding of the FcgammaRIII Receptor by Chimeric Anti-CD20 Antibodies Glyco-Modified by Expression in HEK293-EBNA Nucleic Cells

an acid encoding a ManII-GnTIII fusion polypeptide where the catalytic domain, GnTIII is localized via the ManII localization site in the Golgi complex, where the ManII-GnTIII encoding nucleic acid is expressed either alone (Cbrt antibody) or together with the ManII encoding nucleic acid (Cm ) Cwt is an unmodified recombinant anti-CD20 chimeric IgG1 class C2C8 antibody (“Cwt”) produced by HEK293-EBNA cells transfected with the antibody expression vector pETRT520. Binding analysis was carried out as described in the Materials and Methods section of Example 1. Human NK cells expressing the FcgammaRIIIa receptor on their surface were isolated from the donor organism with a genotype excluding the production of the FcgammaRIIc receptor (i.e., homozygous for the gene variant, which contains an intra-frame stop codon within the sequence encoding FcgammaRIIc). The geometric mean fluorescence intensity, measured by the method of sorting cells with activation of fluorescence (FACS) using fragments of anti-human IgG antibodies labeled with FITC (fluorescein isothiocyanate), increases with increasing number of NK-associated recombinant antibodies. As shown by the use of a competing fragment of an FcgammaRIIIa-specific antibody, the binding determined in this assay is FcgammaRIIIa-specific (See FIG. 13)

FIG. Figure 31: Complement-mediated cytotoxicity of a chimeric anti-CD20 antibody glyco-modified by expression in HEK293-EBNA cells of a nucleic acid encoding a ManII-GnTIII fusion polypeptide, where the catalytic domain, GnTIII is localized via the ManII GII-encoding GII locus, where GIIT encoding the nucleic acid is expressed either alone (Cbrt) or in conjunction with the ManII coding nucleic acid (Cm). Cwt is an unchanged recombinant anti-CD20 chimeric IgG1 class C2C8 antibody ("Cwt") produced by HEK293-EBNA cells transfected with the antibody expression vector pETR1520. Details of the analysis are described in the Materials and Methods section of Example 1.

FIG. 32 (A-C): Expression vectors pCLF9 (A), pETR1842 (B) and pETR1843 (C)

FIG. 33 (A and B): Expression vectors for the fusion proteins ManII-GalT (A) and GalT (B).

FIG. 34: Composition of the oligosaccharides of the anti-CD20 monoclonal antibody produced in the presence of alpha-mannosidase II and the relative percentage of structures associated with the Fc portion of the antibody.

FIG. 35 (A and B): Composition of oligosaccharides of an anti-CD2 monoclonal antibody produced in the presence of the ManII-GalT fusion protein and relative percentage of structures associated with the Fc portion of the antibody. Composition of oligosaccharides after digestion with glycosidases PNGaseF (A) and EndoH (B).

FIG. 36: The antibody produced in the presence of alpha-mannosidase II (ManII) binds the FcγRIIIA receptor with a higher affinity than the wild-type antibody.

FIG. 37: Antibody-dependent cell cytotoxicity mediated by glyco-modified chimeric anti-CD20 antibody.

DETAILED DESCRIPTION OF THE INVENTION

Scientific and technical terms are used here in the meanings commonly used in this field, unless they are additionally defined as follows:

The term "antibody" here includes whole antibody molecules, including monoclonal, polyclonal, and multispecific (e.g., bispecific), as well as antibody fragments having an Fc region, and fusion proteins that contain regions equivalent to the immunoglobulin Fc region. The term also encompasses humanized and chimeric antibodies.

The term "Fc region" here refers to the C-terminal region of the IgG heavy chain. Although the boundaries of the Fc region of the IgG heavy chain may vary slightly, the Fc region of human IgG is usually defined as the region from the amino acid residue at position Cys 226 to the carboxyl end.

The term “region equivalent to the immunoglobulin Fc region” includes here naturally occurring allelic variants of the immunoglobulin Fc region, as well as variants having differences that give rise to substitutions, insertions or deletions, but which do not significantly reduce the ability of the immunoglobulin to mediate effector functions (such as antibody-dependent cellular cytotoxicity). For example, one or more amino acids can be deleted from the N-terminus or C-terminus of the immunoglobulin Fc region without significant loss of biological function. Such options may be selected according to general rules known in the art, so that the effect on activity is minimal. See, e.g., (See, e.g., Bowie, J.U. et al., Science 247: 1306-10 (1990).

The term (recombinant) fusion polypeptide “having beta-1,4-N-acetylglucosaminyltransferase III activity” as used herein refers to fusion polypeptides that are capable of catalyzing the addition of a β-1, -4 bond of an N-acetylglucosamine residue (GlcNAc) to three the remainder of the mannose of the core of the oligosaccharides. This includes fusion polypeptides that exhibit enzymatic activity similar, but not necessarily identical, to that of beta-1,4-N-glucosaminyl transferase III, also known as beta-1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyl transferase (EC 2.4.1.144 according to the nomenclature of NC-IUBMB - the Committee on Nomenclature of the International Union for Biochemistry and Molecular Biology), which is determined by the results of measurements in a special biological analysis (with or without dose dependence). If there is a dose dependence, it does not have to be identical to the dependence inherent in beta-1,4-N-glucosaminyl transferase III (i.e., the test polypeptide can be active more or less than 25 times, preferably less than 10 times less, most preferably less than 3 times less than the activity of beta-1,4-N-acetylglucosaminyl transferase III).

The term (recombinant) fusion polypeptide "having beta-1,4-galactosyltransferase activity" or "having GalT activity" refers to fusion polypeptides that are capable of catalyzing the transfer of a galactose residue from UDP galactose to a non-reducing terminal GlcNAc found in N-linked terminal GlcNAc oligosaccharides. This includes fusion polypeptides that exhibit enzymatic activity similar but not necessarily identical to that of beta-1,4-N-galactosyltransferase, also known as UDF-Gal: N-acetylglucosamine beta-1,4-N-galactosyltransferase (EC 2.4.1.38 according to the nomenclature of NC-IUBMB - the Committee on Nomenclature of the International Union for Biochemistry and Molecular Biology), which is determined by the results of measurements in a special biological analysis (with or without dose dependence). If there is a dose dependence, it does not have to be identical to the inherent beta-1,4-N-galactosyltransferase (i.e., the test polypeptide may be more or less active, no more than 25 times, preferably less not more than 10 times, most preferably less than 3 times less, relative to the activity of beta-1,4-N-galactosyl transferase).

The statement that a nucleic acid or polynucleotide has a nucleotide sequence “identical” to at least, for example, 95% of the reference (reference) reference nucleotide sequence of the present invention means here that the nucleotide sequence or polynucleotide identical to the reference sequence, except that this polynucleotide sequence may include up to five point mutations for every 100 nucleotides of the reference nucleotide sequence. In other words, in order to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to the reference nucleotide sequence, up to 5% of the reference sequence nucleotides can be deleted or replaced with another nucleotide, or a nucleotide insertion of up to 5 can be made % of the total number of nucleotides in the reference sequence. The sequence in question may be the whole sequence shown in FIG. 5 or in FIG. 6.

In practice, whether a certain nucleic acid or polypeptide molecule is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the identical nucleotide sequence or polypeptide sequence of the present invention, it can be determined in the usual way using computer programs known in the art. A preferred method for determining the “best overall match” between the comparison sequence (the sequence of the present invention) and the sequence in question, also known as “complete sequence alignment”, can be carried out using the FASTDB computer program based on the algorithm described in Bratlag et al., Comp. Arr. Biosci. 6: 237-245 (1990). When aligned, both the comparison sequence and the sequence in question are DNA sequences. The RNA sequence can be compared by replacing the nucleotides Y (U) with T (T). The alignment result is expressed as a percentage of identity. The following parameter values are preferred for alignment of DNA sequences using the FASTDB program: Matrix (matrix) = Unitary, k-tuple (maximum match area size) = 4, MismatchPenalty (penalty for mismatch) = 1, Joining Penalty (connection penalty) = 30 , Randomization Group Length (length of the randomization group) = 0, Cutoff Score = 1 (threshold value), Gap Penalty (penalty on introducing gaps (deletions)) = 5, Gap Size Penalty (penalty on gap size) = 0.05, Window Size ( segment size) = 500 or the length of the sequence in question if it is shorter than 500 nucleotides.

If the sequence in question is shorter than the comparison sequence due to deletions at the ends of 3 ′ or 5 ′, and not due to internal deletions, the results should be adjusted “manually”. This is necessary because FASTDB does not consider truncation of 3 'or 5' ends when calculating identity values (%). For the considered sequences truncated compared to the comparison sequence at the 3'- or 5'-end, the value of identity in percent

correct by calculating the percentage of the number of bases of the comparison sequence, which are 3 'or 5' of the considered sequence and which do not match / not aligned, to the total number of bases of the comparison sequence. The match / alignment of the nucleotide is determined by the results of the alignment program FASTDB. The resulting percentage is then subtracted from the identity value (%) previously calculated using the FASTDB program using the specified parameters, and the final identity value is obtained. The adjusted identity value is used for the purposes of this invention. Only bases outside the 3'- and 5'-ends of the sequence in question (which is shown by alignment with the FASTDB program) that do not match / are not aligned with the comparison sequence are taken into account in order to adjust the identity value.

For example, a sequence of 90 bases in length is aligned with a comparison sequence of 100 nucleotides to determine an identity value (%). There are deletions at the 5'-end of the sequence in question, and therefore alignment with FASTDB does not take into account the coincidence / alignment of the first 10 bases at the 5'-end. These 10 unpaired bases account for 10% of the sequence (the number of non-matching bases at the 5'- and 3'-end / total number of bases in the comparison sequence), therefore, 10% is subtracted from the identity value (%) calculated by the FASTDB program. If the remaining 90 bases matched perfectly, the value of identity would be 90%. In another example, a sequence of 90 bases in length is compared with a comparison sequence of 100 bases in length. This time, deletions are internal deletions and, therefore, there are no bases on the 5 'and 3' of the sequence in question that do not match / not align with the comparison sequence. In this case, the percent identity calculated using FASTDB is not manually adjusted. Once again, only the bases [for] 5 ′ and 3 ′ of the considered sequence, which do not match / are not aligned, are subjected to manual correction with the comparison sequence. No other custom correction is acceptable for the purposes of the present invention.

The statement that the polypeptide has an amino acid sequence “identical” to at least, for example, 95% of the comparison amino acid sequence of the present invention, here means that the amino acid sequence of the considered polypeptide is identical to the comparison sequence, except that this considered sequence may include up to five changes amino acids for every 100 amino acids of the amino acid comparison sequence. In other words, in order to obtain a polypeptide having an amino acid sequence at least 95% identical to the amino acid comparison sequence, up to 5% of the amino acid residues of the considered sequence can be inserted, deleted or replaced with another amino acid. These changes with respect to the reference sequence can occur at the N-terminal or C-terminal positions of the amino acid reference sequence or at any positions between these terminal positions, and can be distributed one by one between the residues of the comparison sequence or assembled into one or more continuous groups within the sequence comparisons.

In practice, whether a certain polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the reference polypeptide can be determined in the usual way using computer programs known in the art. . A preferred method for determining the “best overall match” between the comparison sequence (the sequence of the present invention) and the sequence in question, also known as “complete sequence alignment”, can be carried out using the FASTDB computer program based on the algorithm described in Brutlag et al., Comp. Arr. Biosci. 6: 237-245 (1990). When aligning the sequences, the comparison sequence and the sequence in question are simultaneously either nucleotide sequences or amino acid sequences. The results of said “complete sequence alignment” are expressed as a percentage of identity. The following parameters are preferable for alignment of DNA sequences using the FASTDB program: Matrix (matrix) = RAM 0, k-tuple (maximum match area size) = 2, MismatchPenalty (penalty for mismatch) = 1, Joining Penalty (connection penalty) = 20 , Randomization Group Length (length of the randomization group) = 0, Cutoff Score (threshold value) = 1, Gap Penalty (penalty on introducing gaps (deletions)) = 5, Gap Size Penalty (penalty on gap size) = 0.05, Window Size ( segment size) == 500 or the length of the sequence in question, if it is shorter than 500 amino acids.

If the sequence in question is shorter than the comparison sequence due to N-terminal or C-terminal deletions, the results must be adjusted manually. This is necessary because FASTDB does not take into account truncation of the N- or C-terminus when calculating the total value of identity (%). For the sequences under consideration, truncated compared to the comparison sequence at the N- or C-terminus, the identity value (%) is adjusted by calculating the percentage of the number of bases of the comparison sequence, which are the N- or C-ends of the sequence in question and which do not match / not aligned to the total number of bases of the comparison sequence. The match / alignment of the nucleotide is determined by the results of the alignment program FASTDB. The resulting percentage is then subtracted from the identity value (%) calculated previously by the FASTDB program using the specified parameters, and the final identity value (%) is obtained. This final percentage of identity is then used for the purposes of this invention. Only residues at the N- and C-ends of the sequence in question that do not match / are not aligned with the comparison sequence are considered in order to manually adjust the percent identity, that is, only the positions of the residues of the comparison sequence located behind the most final residues of the N- and C-ends the sequence in question.

For example, the considered sequence of 90 amino acid residues in length is aligned with the comparison sequence of 100 residues to determine the identity value (%). There is a deletion at the N-terminus of the sequence in question, and therefore alignment with FASTDB does not show the match / alignment of the first 10 bases at the N-terminus. These 10 unpaired bases account for 10% of the sequence (the number of non-matching bases at the N- and C-ends / total number of bases in the comparison sequence), therefore, 10% is subtracted from the identity value (%) calculated by the FASTDB program. If the remaining 90 bases matched perfectly, the value of identity would be 90%. In another example, a sequence of 90 bases in length is compared with a comparison sequence of 100 bases in length. This time, deletions are internal deletions and therefore, on N and C of the sequence in question there are no bases that do not coincide / are not aligned with the sequence of comparison. In this case, the identity value (%) calculated by the FASTDB program is not manually adjusted. Once again, only the bases beyond the N- and C-ends of the sequence in question (which is shown by alignment with the FASTDB program) that do not match / are not aligned, are manually corrected with the comparison sequence. No other custom correction is acceptable for the purposes of the present invention.

The term "nucleic acid, which" hybridizes under stringent conditions "with the nucleic acid of the present invention, refers to a polynucleotide that hybridizes during an overnight incubation at 42 ° C in a solution containing 50% formamide, 5 × SSC solution (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5 × Denhardt's solution, 10% dextran sulfate and 20 μg / ml denatured digested salmon sperm DNA, followed by washing the filters in 0.1 × SSC at approximately 65 ° C.

The term "domain responsible for localization in the Golgi complex" refers here to the amino acid sequence of a resident polypeptide of the Golgi complex, which is responsible for the "anchoring" of this polypeptide at a position within the Golgi complex. Typically, localization domains are located at the N-terminus of enzymes.

The term "effector function" refers to biological activity that can be attributed to the Fc region (native sequence of the Fc region or variant of the amino acid sequence of the Fc region) of an antibody. Examples of antibody effector functions include without limitation: Fc receptor binding affinity, antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cell phagocytosis (ADCC), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, downregulation of surface receptors, etc.

The terms “modify”, “modified”, “modification” and “glycosylation modification” are considered here to include any manipulations with the pattern (variant of the location of Sugar residues) of glycosylation of naturally occurring polypeptides and their fragments. Glycosylation modification involves the metabolic modification of cell elements involved in glycosylation, including genetic manipulation of oligosaccharide synthesis pathways to achieve altered glycosylation of glycoproteins expressed in cells. In addition, glycosylation modification includes the effect of mutations and the environment surrounding the cell on glycosylation.

The term “host cell” encompasses any cellular system that can be modified to produce modified glycoforms of the desired proteins, protein fragments, or peptides, including antibodies, antibody fragments, and fusion proteins. Typically, the host cell is exposed in such a way as to optimize the expression levels of GnTIII in it. Host cells include cultured cells, for example, cultured mammalian cells such as Chinese hamster ovary (CHO) cells, hamster kidney (KNK) cells, NSO mouse myeloma cells, SP2 / 0 mouse myeloma cells, YO myeloma cells, P3X63 mouse myeloma cells , PER cells, PER cells. C6 or hybridoma cells, yeast cells, insect cells, plant cells, to name just a few, as well as transgenic plant cells, transgenic animals or cultured plant or animal tissues. The term Fc-mediated cell cytotoxicity here includes antibody-dependent cell cytotoxicity mediated by a soluble Fc fusion protein that contains the Fc region of a human antibody. This is an immune mechanism leading to the lysis of “target cells of antibodies” by “effector cells of the human immune system”, where:

“Human immune system effector cells” are a population of leukocytes with Fc receptors on their surface, by which they bind the Fc region of antibodies or Fc fusion proteins and perform an effector function. Such a population may include, but are not limited to, peripheral blood mononuclear cells (PBMCs) and / or natural killer cells (NK cells). “Antibody target cells” are cells bound by antibodies or Fc fusion proteins. Antibodies or Fc fusion proteins bind target cells through the protein portion of the N-terminus of the Fc region.

The term "increased Fc-mediated cell cytotoxicity" is defined here either as an increase in the number of "target cells of antibodies" lysed by the mechanism of Fc-mediated cell cytotoxicity (defined above) for a given time at a given concentration of antibody or Fc protein fusion in the surrounding cell target medium, and / or a decrease in the concentration of antibody or Fc-fusion protein in the environment of the target cell, necessary to achieve lysis by the mechanism of Fc-mediated cell cytotoxicity of a given amount of “glue” antibody current target for a given time. The increase in Fc-mediated cellular cytotoxicity is determined compared to cellular cytotoxicity mediated by the same antibody or Fc-fusion protein produced by the same type of host cells using the same standard methods of preparation, purification and storage, known to specialists in this field, but in the absence of modification of the host cell by the methods described herein for expression of the GnTIII glycosyl transferase. By "antibody having enhanced antibody-dependent cellular cytotoxicity (ADCC)" is meant an antibody having increased ADCC, as determined by any suitable method known to those of ordinary skill in the art. One of the accepted tests for the determination of ADCC in vitro is as follows:

1) in the analysis, target cells are used to expressly express the target antigen recognized by the antigen-binding region of the antibody;

2) in the analysis, human peripheral blood mononuclear cells (MNPCs) obtained from the blood of a randomly selected healthy donor are used as effector cells;

3) the analysis is carried out according to the following protocol

i) PBMCs are isolated using standard density gradient centrifugation procedures and suspended at a concentration of 5 × 10 ⁶ cells / ml in RPMI medium for cell cultures;

ii) target cells are grown using standard tissue culture techniques, harvested in an exponential growth phase (with viability greater than 90%), washed in RPMI medium for cell cultures, labeled with 100 μci ⁵¹ Cr, washed twice with cell culture medium and resuspended in cell medium cultures at a density of 10 ⁶ cells / ml;

iii) transferring 100 μl of the thus prepared final suspension of target cells to each well of a 96-well microplate;

iv) serial dilutions of the antibody from 4000 ng / ml to 0.04 ng / ml in cell culture medium are carried out and 50 μl of the obtained solutions are applied to the target cells in a 96-well microplate, examining different concentrations of the antibody over the entire range indicated above in triplicates.

v) as a control of maximum release (MR), in 3 additional wells on a plate containing labeled target cells, add, instead of the antibody solution (item iv above), 50 μl of a 2% (by volume) aqueous solution of a non-ionic detergent ( Nonidet, Sigma, St. Louis);

vi) as a spontaneous release (SR) control, 50 μl of cell culture RPMI medium are added to 3 additional wells containing labeled target cells, instead of antibody solution (item iv above);

vii) then a 96-well microplate is centrifuged at 50 × g for one minute and incubated for 1 hour at 4 ° C;

viii) 50 μl of an PBMC suspension (item i above) is added to each well of a 96-well microplate (item i above) to obtain a 25: 1 ratio of effector cells to target cells, then the plates are placed in an incubator at 5% atmospheric CO ₂ and incubated at 37 ° C for 1 hour;

ix) harvesting cell free supernatant from each well and experimentally released radioactivity (ER) is determined using a gamma particle counter;

x) for each antibody concentration, the percentage of specific lysis is calculated by the formula: (ER-MR) / (MR-SR) × 100, where ER is the average radioactivity determined (see ix above) for a given concentration of antibody, MR - the average radioactivity determined for the maximum release controls, paragraph v above), and SR is the average radioactivity determined for the spontaneous release controls (see paragraph vi above);

4) the term "increased ADCC" is defined either as an increase in the maximum percentage of specific lysis observed in the concentration range of the antibodies studied as described above, and / or a decrease in the concentration of antibody necessary to achieve half the maximum percentage of specific lysis observed in the concentration range antibodies tested as described above. An increase in ADCC is determined relative to that determined using the ADCC assay described above, mediated by the same antibody produced by the host cells of the same type, using the same standard methods of preparation, purification and storage, known to those skilled in the art, but which were not produced by the cell a host modified by the methods described herein for expressing a GnT III glycosyltransferase.

The term anti-CD20 antibody is used here to refer to an antibody that specifically recognizes a 35,000 Da non-glycosylated phosphoprotein located on the surface of a cell, commonly referred to as the Bp35 human narrowly differentiated B lymphocyte antigen, often referred to as CD20.

The present invention is based on the discovery that antibody-producing cells are modified to express a new fusion protein having beta-1,4-N-acetylglucosaminyl transferase III (GnTIII) activity or, alternatively, beta-1,4-N-galactosyl transferase activity (GalT) and containing the Golgi complex resident polypeptide domain responsible for localization in the Golgi complex, results in antibodies with increased Fc receptor binding affinity and increased effector function. Alternatively, by modifying antibody producing cells for increased expression of a nucleic acid molecule encoding a polypeptide having catalytic activity of mannosidase II, antibodies with enhanced effector function and / or Fc receptor binding can be obtained. In preferred embodiments, fusion constructs having GnTIII or GalT activity are expressed together with nucleic acid molecules encoding ManII or GnTII.

Preferably, the isolated nucleic acid has the nucleotide sequence shown in FIG. 24 and SEQ ID NO: 12. In another preferred embodiment, the fusion polypeptide comprises a catalytic domain of beta-1,4-N-acetylglucosaminyl transferase III, and the localization site in the Golgi complex is a domain of beta-1,2-N-acetylglucosaminyl transferase I (GnTI ) Preferably, the nucleic acid has the nucleotide sequence shown in FIG. 25 and SEQ ID NO: 14. Alternatively, the domain of another Golgi complex resident polypeptide responsible for localization in the Golgi complex can be used. In another preferred embodiment, the Golgi complex localization domain is selected from the group consisting of the beta-1,2-N-acetylglucosaminyl transferase II localization domain, the mannosidase I localization domain, and the alpha1-6 corefucosyl transferase localization domain.

In another preferred embodiment, the present invention provides an isolated nucleic acid comprising a sequence that encodes a polypeptide having the amino acid sequence shown in FIG. 24 and SEQ ID NO: 13 or in FIG. 25 and SEQ ID NO: 15. The present invention also includes an isolated nucleic acid that hybridizes under stringent conditions to a hybridization probe with a nucleotide sequence that consists of the nucleotide sequence shown in FIG. 24 and SEQ ID NO: 12 or FIG. 25 and SEQ ID NO: 14. The invention further provides an isolated nucleic acid comprising a sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence shown in FIG. 24 and SEQ ID NO: 12 or FIG. 25 and SEQ ID NO: 14. In another embodiment, the invention provides an isolated nucleic acid comprising a sequence encoding a polypeptide having an amino acid sequence of at least 80%, 85%, 90%, 95%, 96%, 97%, 98 % or 99% identical to the amino acid sequence shown in FIG. 24 and SEQ ID NO: 13 or FIG. 25 and SEQ ID NO: 15. The invention also includes an isolated nucleic acid comprising a sequence that encodes a polypeptide having the amino acid sequence of FIG. 24 and SEQ ID NO: 13 or FIG. 25 and SEQ ID NO: 15 with conservative amino acid substitutions.

The present invention further provides a method for modifying the glycosylation profile of a polypeptide produced by a host cell, comprising introducing into the specified host cell a nucleic acid molecule or vector of the present invention. Preferably, the modified polypeptide is IgG or a fragment thereof containing an Fc region. In another preferred embodiment, the modified polypeptide is a fusion protein that includes a region equivalent to the Fc region of human IgG.

The present invention further provides a host cell comprising a nucleic acid and an expression vector of the present invention. In one embodiment, the present invention provides a host cell modified to express at least one nucleic acid encoding a fusion polypeptide having beta-1,4-N-acetylglucosaminyl transferase III activity, or, alternatively, beta-1,4- activity galactosyl transferase ("GalT"), in an amount sufficient to modify the oligosaccharides in the Fc region of the polypeptide produced by the specified host cell, where the specified polypeptide is selected from the group consisting of the whole antibody molecule, fragment coagulant antibody containing Fc-fragment and the fusion protein which comprises a region equivalent to the Fc-region of an immunoglobulin. In a preferred embodiment, the polypeptide produced by said host cell is IgG or a fragment thereof. Most preferably, the polypeptide produced by said host cell is IgG1 or a fragment thereof. Alternatively, the polypeptide produced by said host cell is a protein that contains a region equivalent to the Fc region of human IgG, for example, IgG1.

The modified polypeptides produced by the host cell of the present invention, as a result of the modification, exhibit increased Fc receptor binding affinity and / or increased effector function. Preferably, the increased binding affinity of the Fc receptor is increased binding of the activating Fcγ receptor, such as the FcγRIIIa receptor. Increased effector function is preferably an increase in one or more of the following functions: increased antibody-dependent cellular cytotoxicity, increased antibody-dependent cell phagocytosis (ASCF), increased cytokine secretion, increased immune complex mediated antigen uptake by antigen-presenting cells, increased Fc-mediated, increased cell cytotoxicity binding to NK cells, increased binding to macrophages, increased binding to polymorphonuclear cells, increased monocyte binding, increased cross-linking with antibodies bound to (other) targets, increased direct transmission of apoptosis-inducing signals, increased dendritic cell maturation, increased T-cell priming activity (differentiation induction).

In a particularly preferred embodiment, the host cell of the present invention is a CHO cell, BHK cell, NSO cell, SP2 / 0 cell, YO myeloma cell, P3X63 mouse myeloma cell, PER cell, PER.C6 cell or hybridoma cell, and the polypeptide produced by said the host cell is an anti-CD20 antibody, such as IDEC-C2B8. In another preferred invention, the host cell is a C225 antibody against human EGFR (epidermal growth factor receptor).

In addition to the nucleic acid encoding the fusion polypeptide of the invention, the host cell of the invention may comprise at least one nucleic acid fragment encoding an antibody molecule that retains its functional Fc region, an antibody fragment or fusion protein that contains a region equivalent to Fc- areas of immunoglobulin. In preferred embodiments, this at least one nucleic acid segment encodes an anti-CD20 antibody, a chimeric anti-human neuroblastoma chCE7 monoclonal antibody, a chG250 anti-renal cell carcinoma monoclonal antibody, an ING-1 chimeric anti-colon, lung and breast carcinoma monoclonal antibody, humanized 3622W94 against human antigen 17-1A, humanized A33 antibody against colorectal tumor, R24 anti-human melanoma antibody directed to GD3 ganglioside, x three-dimensional monoclonal antibody SF-25 against human squamous cell carcinoma, antibody against human REFR, antibody against receptor for epidermal growth factor human REFR vIII, antibody against PSMA (prostate-specific membrane antigen), antibody against surface stem cell antigen of the prostate (PSCA), antibody anti-human CD22, anti-human CD30 antibody, anti-high molecular weight tumor associated human melanoma antigen (HMWMAA), anti-ganglioside GD3 antibody, anti-gD ganglioside antibody, anti anti-ganglioside antibody GM2, anti-human ganglioside antibody, anti-human epidermal growth factor antibody EGFRvIII (EGFR) antibody, anti-integrin antibody, anti-CDSO antibody, anti-LeY antibody, anti-mucin antibody, anti-MUC18 antibody, anti-CD33 antibody human, anti-human CD38 antibody, anti-human CD40 antibody, human anti-CD45 antibody, human anti-CD52 antibody, human anti-CD 138 antibody, anti-HLA antibody (HLA-DR variant), anti-human EpCAM antibody, anti-human CEA antibody, anti-human antibody MUC1 protein eloveka, bark MUC1 antibody against human protein, the antibody against human aberrant glycosylated MUC1 protein antibody containing ED-B domain of human fibronectin variants or anti-HER2 / neu human.

The present invention also provides a method for producing a polypeptide in a host cell, comprising (a) culturing a host cell modified to express at least one nucleic acid encoding a fusion polypeptide having beta-1,4-N-acetylglucosaminyl transferase III activity, or, alternatively, beta-1,4-galactosyltransferase (“GalT”) activity, under conditions that allow the cell to produce a polypeptide selected from the group consisting of an entire antibody molecule, an antibody fragment that retains the functional Fc region and a fusion protein that contains a region equivalent to the immunoglobulin Fc region, wherein said polypeptide having GnTII activity or, alternatively, GalT activity is expressed in an amount sufficient to modify the oligosaccharides of the Fc region of said polypeptide produced by the host cell; and (b) isolating said polypeptide. In a preferred embodiment, the fusion polypeptide comprises a beta-1,4-N-acetylglucosaminyl transferase catalytic domain. In a particularly preferred embodiment, the fusion protein also contains the localization in the Golgi complex domain of any resident polypeptide of the Golgi complex. Preferably, the localization domain in the Golgi complex is the localization domain of mannosidase II or beta-1,2-N-acetylglucosaminyl transferase I (GnTI). Alternatively, the domain responsible for localization in the Golgi complex is selected from the group consisting of the localization domain of mannosidase I, the localization domain of beta-1,2-N-acetylglucosaminyl transferase II, and the localization domain alpha1-6 of corfucosyl transferase. Polypeptides produced by the methods of the present invention have enhanced Fc receptor binding affinity and / or enhanced effector function. Increased effector function is preferably an increase in one or more of the following functions: Fc-mediated cell cytotoxicity (including increased antibody-dependent cellular cytotoxicity), increased antibody-dependent cell phagocytosis (ASCP), increased cytokine secretion, increased immune complex mediated antigen-uptake antigen-present antigen-absorption increased Fc-mediated cell cytotoxicity, increased binding to NK cells, increased binding to ma rofagami, increased binding to monocytes, increased binding to polymorphonuclear cells, increased crosslinking with related (other) target antibodies, increased direct transmission signals inducing apoptosis, increased dendritic cell maturation, the increased activity of T lymphocyte priming. The increased binding affinity of the Fc receptor is preferably an increased binding of the activating Fcγ receptor, such as the FcγRIIIa receptor.

In another embodiment, the present invention provides a polypeptide produced by the methods of the present invention, in which the proportion of branched oligosaccharides in the Fc region of the polypeptide is increased. In another embodiment, the polypeptide produced by the methods of the invention has, as a result of this modification, an increased proportion of unfucosylated oligosaccharides in its Fc fragment. Said non-fucosylated oligosaccharides may be hybrid or complex type oligosaccharides. In a particularly preferred embodiment, the polypeptide produced by the host cell and by the methods of the present invention has an increased proportion of bisected non-fucosylated oligosaccharides in the Fc region. Branched non-fucosylated oligosaccharides can be either hybrid or complex. In particular, the methods of the present invention can be used to produce polypeptides in which at least 15%, preferably at least 20%, more preferably at least 25%, even more preferably at least 30%), more preferably at least 35%, even more preferably at least 40%, even more preferably at least 45%, even more preferably at least 50%, even more preferably at least 55%, still more preferably at least 60%, even more e preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, still more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99% of the oligosaccharides in the Fc region of the polypeptide are non-fucosylated oligosaccharides. The methods of the present invention can also be used to produce polypeptides in which at least 15%, more preferably at least 20%, even more preferably at least 25%, even more preferably at least 30%, even more preferably at least 35%, even more preferably at least 40%, even more preferably at least 45%, even more preferably at least 50%, even more preferably at least 55%, even more preferably at least 60%, still b more preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, still more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99% of the oligosaccharides in the Fc region of the polypeptide are bisected oligosaccharides. Further, the methods of the present invention can be used to produce polypeptides in which at least 15%, more preferably at least 20%, even more preferably at least 25%, even more preferably at least 30%, still more preferably at least 35%, even more preferably at least 40%, even more preferably at least 45%, even more preferably at least 50%, even more preferably at least 55%, still more preferably at least 60%, still b more preferably at least 70%, even more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, even more preferably at least 95%, still more preferably at least 96%, even more preferably at least 97%, even more preferably at least 98%, even more preferably at least 99% of the oligosaccharides in the Fc region of the polypeptide are bisected , unfucosylated oligosaccharides. The methods of the present invention can also be used to produce polypeptides in which at least 15%, more preferably at least 20%, even more preferably at least 25%, even more preferably at least 30%, even more preferably, at least 35% of the oligosaccharides in the Fc region of the polypeptide are branched hybrid non-fucosylated oligosaccharides.

In another embodiment, the present invention provides an antibody modified to increase effector function and / or increase the binding affinity of the Fc receptor. Preferably, increased effector function is one of the following: increased Fc-mediated cell cytotoxicity (including increased antibody-dependent cellular cytotoxicity), increased antibody-dependent cell phagocytosis (ASCF), increased cytokine secretion, increased immune complex mediated antigen uptake, presenting antigen presenting NK cells, increased binding to macrophages, increased binding to monocytes, increased polymorphonuclear binding GOVERNMENTAL cells, increased crosslinking with related (other) target antibodies, increased direct transmission signals inducing apoptosis, increased dendritic cell maturation, the increased activity of priming T-cells (induction of differentiation). In a preferred embodiment, the increased binding affinity of the Fc receptor is increased binding of the activating Fcγ receptor, most preferably to the FcγRIIIa receptor. The invention further provides antibody fragments containing the Fc region and fusion proteins that contain a region equivalent to the immunoglobulin Fc region. Such antibody fragments and fusion proteins exhibit increased Fc receptor binding affinity and / or increased effector function.

The present invention further provides pharmaceutical formulations containing antibodies that retain the Fc region of antibody fragments and fusion proteins having a region equivalent to the immunoglobulin Fc region of the present invention and a pharmaceutically acceptable carrier.

The present invention further provides the use of such pharmaceutical compositions in a method of treating cancer. In particular, the present invention provides a method for treating cancer, comprising administering a therapeutically effective amount of the pharmaceutical compositions of this invention.

The present invention also provides a host cell containing an expression vector containing a nucleic acid molecule encoding a fusion polypeptide, wherein said polypeptide has beta-1,4-N-acetylglucosaminyl transferase III (GnTIII) activity and contains any localization in the Golgi complex a resident protein of the Golgi complex, as well as an expression vector containing a nucleic acid molecule encoding a polypeptide, where the polypeptide has mannosidase II (ManII) activity. In preferred embodiments, the nucleic acid molecule encoding a fusion polypeptide and the nucleic acid molecule encoding a polypeptide having mannosidase I activity are in one or in separate expression vectors. In another preferred embodiment, the fusion polypeptide comprises a GnTIII catalytic domain. In a further preferred invention, the Golgi complex localization domain is the localization domain of ManII, beta-1,2-N-acetylglucosaminyl transferase II, mannosidase I, or alpha 1,6-N corefucosyl transferase.

In a further preferred embodiment, the host cell is selected from the group consisting of a yeast cell, an insect cell or a plant cell. The invention further provides a host cell containing an expression vector containing a nucleic acid molecule encoding a fusion polypeptide having beta-1,4-N-acetylglucosaminyl transferase III (GnTIII) activity and containing the domain of any resident protein responsible for localization in the Golgi complex Golgi complex, an expression vector containing a nucleic acid molecule encoding a polypeptide that has mannosidase II (ManII) activity, and an expression vector containing a nucleic acid molecule encoding a polypeptide, wherein the polypeptide has beta-1,2-N-acetylglucosaminyl transferase II (GnTII) activity. In preferred embodiments, the nucleic acid molecule encoding the fusion polypeptide, the nucleic acid molecule encoding the polypeptide having ManII activity, and the nucleic acid molecule encoding a polypeptide having GnTII activity are in one or in separate expression vectors. It is also preferred that the nucleic acid molecule encoding the fusion polypeptide is in one expression vector, and the nucleic acid molecule encoding the polypeptide having ManII activity and the nucleic acid molecule encoding the polypeptide having GnTII in another expression vector. It is also preferable that the nucleic acid molecule encoding ManII is in one expression vector, and the nucleic acid molecule encoding a fusion polypeptide and the nucleic acid molecule encoding a polypeptide having GnTII activity in another expression vector. In another embodiment, GnTII is in one expression vector, and a nucleic acid molecule encoding a fusion polypeptide and a nucleic acid molecule encoding a polypeptide having ManII activity is another expression vector.

In an additional aspect, the invention provides a host cell containing an expression vector containing a nucleic acid molecule encoding a fusion polypeptide, where the fusion polypeptide has beta-1,4-N-galactosyl transferase (GalT) activity and is responsible for the localization of any Golgi complex a Golgi complex resident polypeptide, as well as an expression vector containing a nucleic acid molecule encoding a polypeptide, where the polypeptide has mannosidase II (ManII) activity. In preferred embodiments, the nucleic acid molecule encoding a fusion polypeptide and the nucleic acid molecule encoding a polypeptide having mannosidase P activity are in one or in separate expression vectors. Preferably, the fusion polypeptide contains a GalT catalytic domain. In a further embodiment, the localization domain in the Golgi complex is the localization domain of ManII, beta-1,2-N-acetylglucosaminyl transferase I, beta-1,2-N-acetylglucosaminyl transferase II, mannosidase I, or alpha 1,6-N corfucosyl transferase . In a preferred embodiment, the host cell is selected from the group consisting of a mammalian cell, a yeast cell, an insect cell, or a plant cell. Preferably, the host cell is a CHO cell, an BHK cell, an NSO cell, an SP2 / 0 cell, a YO myeloma cell, a P3X63 mouse myeloma cell, a PER cell, a PER cell, a PER cell. C6 or hybridoma cell.

In an additional aspect, the invention provides a host cell containing an expression vector containing a nucleic acid molecule encoding a fusion polypeptide, where the fusion polypeptide has beta-1,4-N-galactosyltransferase (GalT) activity and contains a domain which is responsible for the localization in the Golgi complex or a resident protein of the Golgi complex, as well as an expression vector containing a nucleic acid molecule encoding a polypeptide, where the polypeptide has mannosidase II (ManII) activity, and an expression vector containing th nucleic acid molecule encoding a polypeptide which has an activity of beta-1,2-N-atsetilglyukozaminiltransferazy II (GnTII). Preferably, all nucleic acids are in the same expression vector. In another embodiment of the invention, each of the nucleic acid molecules is in a separate expression vector. The invention further provides that a nucleic acid molecule encoding a fusion polypeptide is in one expression vector, and a nucleic acid molecule encoding a polypeptide having ManII activity, and a nucleic acid molecule encoding a polypeptide having GnTII activity, is another expression vector. The invention also provides that the nucleic acid molecule encoding ManII is in one expression vector, and the nucleic acid molecule encoding a fusion polypeptide, and the nucleic acid molecule encoding a polypeptide having GnTII activity in another expression vector. The invention also provides that the nucleic acid molecule encoding GnTII is in one expression vector, and the nucleic acid molecule encoding the fusion polypeptide, and the nucleic acid molecule encoding a polypeptide having ManII activity in another expression vector. Further, in a preferred embodiment, the localization domain in the Golgi complex is the localization domain of ManII, beta-1,2-N-acetylglucosaminyl transferase I, beta-1,2-N-acetylglucosaminyl transferase II, mannosidase I, or alpha 1,6-N cor- fucosyl transferase.

The invention further provides a host cell modified to express at least one nucleic acid encoding a fusion polypeptide having GnTIII activity, and at least one nucleic acid encoding a fusion polypeptide having ManII activity, in an amount sufficient to modify the oligosaccharides in Fc- the region of the polypeptide produced by the host cell, where the polypeptide produced by the host cell is selected from the group consisting of a whole antibody molecule, an antibody fragment and a fusion protein, ory contains a region equivalent to an immunoglobulin Fc-region.

The invention also provides a host cell modified to express at least one nucleic acid encoding a fusion polypeptide having GnTIII activity, at least one nucleic acid encoding a polypeptide having ManII activity, and at least one nucleic acid encoding a polypeptide, possessing GnII activity in an amount sufficient to modify the oligosaccharides in the Fc region of the polypeptide produced by the host cell, where the polypeptide produced by the host cell irayut from the group consisting of a whole antibody molecule, an antibody fragment, and fusion protein which comprises a region equivalent to the Fc-region of an immunoglobulin.

The present invention further provides a host cell modified to express at least one nucleic acid encoding a fusion polypeptide having GalT activity, and at least one nucleic acid encoding a polypeptide having ManII activity, in an amount sufficient to modify the oligosaccharides in Fc -regions of the polypeptide produced by the host cell, where the polypeptide produced by the host cell is selected from the group consisting of a whole antibody molecule, an antibody fragment, and a fusion protein that contains a region equivalent to the immunoglobulin Fc region.

In a separate embodiment, the present invention also provides a host cell modified to express at least one nucleic acid encoding a fusion polypeptide having GalT activity, at least one nucleic acid encoding a polypeptide having ManII activity, and at least one nucleic acid encoding a polypeptide having GnIIT activity in an amount sufficient to modify oligosaccharides in the Fc region of the polypeptide produced by the host cell, where the polypeptide, pr induced by the host cell is selected from the group consisting of a whole antibody molecule, an antibody fragment, and a fusion protein that contains a region equivalent to the immunoglobulin Fc region. Preferably, the polypeptide produced by the host cell exhibits, as a result of the modification, an increased binding affinity of the Fc receptor. In a further preferred embodiment of the invention, the polypeptide produced by the host cell exhibits, as a result of the modification, increased effector function. Preferably, increased effector function is one or more of the following: increased Fc-mediated cell cytotoxicity; increased binding to NK cells; increased binding to macrophages; increased binding to polymorphonuclear cells; increased binding to monocytes; increased cross-linking to bound to (other) targets with antibodies, increased direct transmission of apoptosis-inducing signals, increased maturation of dendritic cells and / or increased activity of T-priming letok (induction of differentiation).

In a further embodiment, the invention provides a method for producing a polypeptide in a host cell, comprising culturing a host cell modified to express at least one nucleic acid encoding a fusion polypeptide having GnTIII activity and at least one nucleic acid encoding a polypeptide having activity ManII, under conditions that allow the production of a polypeptide selected from the group consisting of a whole antibody molecule, an antibody fragment and a fusion protein, cat ing comprises a region equivalent to an immunoglobulin Fc-region where fusion polypeptide is expressed in an amount sufficient to modify the oligosaccharides in the Fc-region of a polypeptide produced by the host cell and the isolation of the polypeptide. Preferably, the host cell is also modified to express at least one nucleic acid encoding a polypeptide having GnTII activity. In a further preferred embodiment, the fusion polypeptide comprises a GnTIII catalytic domain. In a further preferred embodiment, the fusion polypeptide also contains the domain of a heterologous resident Golgi complex polypeptide responsible for localization in the Golgi complex. Preferably, the localization domain in the Golgi complex is the localization domain of mannosidase II, beta-1,2-N-acetylglucosaminyl transferase I, mannosidase I, beta-1,2-N-acetylglucosaminyl transferase II or alpha 1,6-N corefucosyl transferase . Preferably, the polypeptide has, as a result of the modification described above, increased effector function.

The invention further provides a method for producing a polypeptide in a host cell, comprising culturing a host cell modified to express at least one nucleic acid encoding a fusion polypeptide having GalT activity, and at least one nucleic acid encoding a ManII polypeptide having activity conditions that allow the production of a polypeptide selected from the group consisting of a whole antibody molecule, an antibody fragment and a fusion protein that contains a region, equivalent to the immunoglobulin Fc region, where the fusion polypeptide is expressed in an amount sufficient to modify the oligosaccharides in the Fc region of the polypeptide produced by the host cell, as well as the isolation of this polypeptide. In a further embodiment, the host cell is also modified to express at least one nucleic acid encoding a polypeptide having GnTII activity. Preferably, the fusion polypeptide contains a GalT catalytic domain. Also preferably, the fusion polypeptide also contains the domain of a heterologous Golgi complex resident polypeptide responsible for localization in the Golgi complex. Preferably, the localization domain in the Golgi complex is the localization domain of mannosidase II, beta-1,2-N-acetylglucosaminyl transferase I, mannosidase I, beta-1,2-N-acetylglucosaminyl transferase II or alpha 1,6-N corefucosyl transferase . Preferably, the polypeptide has, as a result of the modification described above, increased effector function. Specifically, in a preferred embodiment, the polypeptide produced by the host cell has an increased proportion of bisected non-fucosylated oligosaccharides in the Fc region of the polypeptide. Preferably, branched non-fucosylated oligosaccharides are hybrid. Even more preferably, branched non-fucosylated are complex. In a preferred embodiment, at least about 10% to 95% of the oligosaccharides in the Fc region of the polypeptide are branched unfucosylated. It is particularly preferred that approximately 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the oligosaccharides of the Fc region of the glyco-modified polypeptides of the present invention were branched and non-fucosylated oligosaccharides.

In a further preferred embodiment, the present invention provides that antibodies modified according to the methods of the present invention have enhanced effector function.

In a further embodiment, the invention further provides pharmaceutical compositions comprising antibodies modified according to the methods of the present invention. Preferably, the pharmaceutical compositions of this invention contain a pharmaceutically acceptable carrier.

The invention further provides a method for treating cancerous tumors, comprising administering a therapeutically effective amount of a pharmaceutical composition according to the present invention to a patient in need of treatment. The invention further provides a method for producing a polypeptide having increased Fc-mediated cell cytotoxicity in a host cell, comprising culturing a host cell modified to express at least one nucleic acid encoding GalT and at least one nucleic acid encoding ManII in conditions that allow the production of a polypeptide selected from the group consisting of a whole antibody molecule and a fragment of an antibody that contains the immunoglobulin Fc region, where ur the degree of expression of one of the GalT and ManII polypeptides, or both, is sufficient to modify the oligosaccharides in the Fc region of the polypeptide produced by the host cell, and where the polypeptide has, as a result of the modification, increased Fc-mediated cell cytotoxicity, as well as the isolation of a polypeptide having increased Fc-mediated cell cytotoxicity. In a further preferred embodiment of the invention, the level of GalT expression leads to the appearance of an antibody molecule or antibody fragment that contains an immunoglobulin Fc region having increased Fc-mediated cell cytotoxicity.

The invention further provides a method for producing a polypeptide having increased Fc-mediated cell cytotoxicity in a host cell, comprising culturing a host cell modified to express at least one nucleic acid encoding GalT and at least one nucleic acid encoding ManII, under conditions that allow the production of a polypeptide selected from the group consisting of a whole antibody molecule, an antibody fragment that contains the immunoglobulin Fc region, where the lack of expression of one of the GalT and ManII polypeptides, or both of them, is sufficient to modify the oligosaccharides in the Fc region of the polypeptide produced by the host cell, and where the polypeptide has, as a result of the modification, increased Fc-mediated cell cytotoxicity, as well as the isolation of a polypeptide having increased Fc-mediated cell cytotoxicity. In a preferred embodiment, the host cell described above also contains at least one nucleic acid encoding GnTIII, where GnTIII is expressed in an amount sufficient to modify the oligosaccharides in the Fc region of the polypeptide produced by the host cell, where the polypeptide has, as a result of the modification, increased Fc-mediated cell cytotoxicity. Preferably, the level of expression of one or more of the GalT, ManII, and GnTIII polypeptides is sufficient to form branched (bisected) oligosaccharides in the Fc region of the polypeptide. Even more preferably, the proportion of branched [oligosaccharides in the Fc region of the polypeptide is at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 percent. Preferably, the proportion of branched [oligosaccharides in the Fc region is at least 45 percent. In a preferred embodiment, the branched oligosaccharides are hybrid. Preferably, the host cell is a mammalian cell, a yeast cell or a plant cell. Even more preferably, the host cell is a plant cell.

In another aspect, the present invention provides a method for producing a polypeptide by a host cell, comprising:

a. culturing a host cell modified to express at least one nucleic acid encoding a polypeptide having alpha-mannosidase II activity under conditions that allow for the production of a polypeptide selected from the group consisting of a whole antibody molecule, an antibody fragment and a fusion protein that contains a region equivalent to the immunoglobulin Fc region, wherein said polypeptide having alpha-mannosidase II activity is expressed in an amount sufficient to modify the oligosaccharides in the Fc region of said polypeptide produced by the host cell; and

b. Isolation of the indicated polypeptide produced by the specified host cell.

The present invention also provides polypeptides, in particular antibodies, having, as a result of said modification of oligosaccharides, enhanced effector function and / or increased binding of the Fc receptor, as well as their use in therapeutic compositions for treating disorders, in particular tumors.

Identification and preparation of nucleic acids encoding a protein whose glycosylation pattern is desired to be modified.

The present invention provides methods for producing and using host-cell systems to produce antibody glycoforms, antibody fragments, and fusion proteins that contain a region equivalent to the immunoglobulin Fc region having increased binding affinity of the Fc receptor (preferably an activating Fc receptor) and / or increased effector functions, including antibody-dependent cellular cytotoxicity. The identification of target epitopes and the creation of antibodies of possible therapeutic value, the glycosylation pattern of which it is desirable to modify, as well as the isolation of the corresponding nucleic acid coding sequences are within the scope of the invention.

A variety of procedures known in the art can be used to produce antibodies to target epitopes. Such antibodies include, without limitation: polyclonal, monoclonal, chimeric, humanized, fully human and single chain antibodies, Fab fragments and fragments obtained from the ScFv, Fab, VH, IgG expression library. Such antibodies may be useful as, for example, diagnostic or therapeutic agents. Neutralizing antibodies, i.e. those antibodies that compete for binding to a ligand, substrate or adapter molecule are of particular interest as therapeutic agents.

To produce antibodies, a variety of animals (hosts), including, but not limited to, rabbits, mice, rats, etc., are immunized by injection with the target protein. A large number of polyclonal antibodies can be obtained by multiple subcutaneous (pc) or intraperitoneal (wb) injections of the corresponding antigen and adjuvant. To enhance the immunological response, various adjuvants can be used, depending on the type of host animal, including but not limited to Freund's adjuvant (complete and incomplete), mineral gels, such as aluminum hydroxide, surfactants, such as lysolecithin, polyalcohols pluronic, polyanions, proteins, saponin, oil emulsions, saucer hemocyanin (KLH, GMB), dinitrophenol and potentially useful human adjuvants such as BCG (bacilli Calmette-Guerin) and Corinobacterium parvum. Animals are immunized against antigen, immunogenic conjugates or derivatives by mixing, for example, 100 g or 5 g of protein or conjugate (for a rabbit or mouse, respectively) with three volumes of Freund's complete adjuvant and subsequent administration of the solution by subcutaneous injection at multiple sites. After a month, supportive immunization is carried out by injection at multiple sites from 1/5 to 1/10 of the original volume of the protein or conjugate in Freund's adjuvant. After 7-14 days, blood is collected from the animals and the antibody titer is determined. Supporting immunizations are carried out until the titer stops increasing. Preferably, the supporting immunizations of the animal are carried out with the same antigen, but conjugated to another protein and / or via another binding substance. Monoclonal antibodies to the target can be prepared using any technology that involves the production of antibody molecules by continuous cell lines in culture. Such technologies include, without limitation: the hybridoma technology first described in Kohler, Milstein, Nature 256: 495-97 (1975), the hybridoma human B cell technology (Kosbor et al., Immunology Today 4: 72 (1983); Cote et al ., Proc. Natl. Acad. Set USA 80: 2026-30 (1983) and EBV hybridoma technology (Cole et al, Monoclonal Antibodies and Cancer Therapy 77-96 (Alan R. Liss, Inc., 1985)). an addition may involve technology for producing “chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. USA 81: 6851-55 (1984); Neuberger et al., Nature 312: 604-08 (1984); Takeda et al., Nature 314: 452-54 (1985) by splicing (linking) the genes of a mouse antibody molecule of the corresponding antigenic specificity with genes of a human antibody molecule with suitable biological activity.This technology can also be used to obtain chimeric antibodies composed of antibody molecules of other mammals.Alternatively, single-chain antibody production technology can be adapted to produce single chain antibodies of the desired specificity (US Patent No. 4,946, 778). The invention further provides humanized antibodies subjected to glycomodification according to the methods of the present invention. Technologies for creating humanized antibodies are disclosed, for example, in U.S. Patent U.S. Pat. No. 6,180, 320 to Queen et al, the entire contents of which are incorporated herein by reference in their entirety.

Antibody fragments that contain specific binding sites of a target protein can be obtained by known techniques. For example, such fragments include, without limitation: F (ab ') ₂ fragments, which can be obtained by pepsin cleavage of the antibody molecule, and Fab fragments, which can be obtained by destroying the disulfide bridges of F (ab') ₂ fragments. Alternatively, Fab expression libraries can be created (Huse et al., Science 246: 1275-81 (1989), which will quickly and easily identify monoclonal Fab fragments of the desired specificity for the target protein.

After identifying the antibody or antibody fragment whose glycosylation pattern it is desired to modify, the nucleic acid coding sequence is identified and isolated, for which technology well known in the art is used.

but. Obtaining cell lines for the production of proteins with an altered glycosylation pattern (pattern - variant, pattern).

The present invention provides host cell expression systems for producing proteins with a modified glycosylation pattern. In particular, the present invention provides a host cell system for producing glycoform proteins of enhanced therapeutic value. Therefore, in one aspect, the invention provides host-cell expression systems selected or modified for expression of, for example, a fusion protein having beta-1,4-N-acetylglucosaminyl transferase III (GnTIII) activity and containing a domain which is responsible for localization in the Golgi complex or a heterologous Golgi complex resident polypeptide. In particular, such host-cell expression systems may be modified with a recombinant nucleic acid molecule encoding such a fusion protein operably linked to a constitutive or regulated promoter system.

In one particular embodiment, the present invention provides a host cell that has been modified to express at least one nucleic acid encoding a fusion protein having beta-1,4-N-acetylglucosaminyl transferase III (GnTIII) activity and containing complex localization Golgi is the domain of any heterologous polypeptide resident in the Golgi complex. In one aspect, the host cell is modified with a nucleic acid molecule containing at least one gene encoding a fusion polypeptide having beta-1,4-N-acetylglucosaminyl transferase III (GnTIII) activity and containing a heterologous domain for localization in the Golgi complex Golgi complex resident polypeptide.

In general, any type of cultured cell line can be used as the basis for constructing cell lines according to the present invention. In a preferred embodiment, yeast cells, insect cells or plant cells are used as the basis for the preparation of the modified host cells of the present invention. (See Ma, J. K.-C. et al., Nature Genetics 4: 794-805 (October 2003) and references cited therein) (the entire contents of which are incorporated herein by reference).

The invention is intended to include modified host cells expressing a fusion polypeptide having beta-1,4-N-acetylglucosaminyl transferase III (GnTIII) activity and containing the domain of any heterologous Golgi complex resident polypeptide responsible for localization in the Golgi complex.

One or more amino acids encoding a fusion polypeptide having beta-1,4-N-acetylglucosaminyl transferase III (GnTIII) activity and containing the domain of a heterologous Golgi complex heterologous polypeptide responsible for localization in the Golgi complex can be expressed under the control of a constitutive promoter or , as an alternative, a regulated expression system. Suitable regulated expression systems include, but are not limited to: a tetracycline-regulated expression system, an ecdysone-induced expression system, an expression system comprising a lac promoter, a glucocorticoid-induced expression system, a temperature-induced promoter system, and a metallothionine metal-induced expression system. If the host-cell system contains several nucleic acid molecules encoding fusion proteins with beta-1,4-N-acetylglucosaminyl transferase III (GnTIII) activity and containing the domain of a heterologous resident polypeptide of the complex, which is responsible for localization in the Golgi complex Golgi, some of them can be expressed under the control of a constitutive promoter, while others are expressed under the control of a regulated promoter. By maximum expression level is meant the highest possible level of stable expression of a fusion protein that does not have a significant negative effect on cell growth rate. The maximum level of expression can be determined during routine experiments. Expression levels are determined by methods well known in the art, including Western blot using an antibody specific for a protein having GnTIII activity, or an antibody specific for a protein label site attached to a polypeptide having GnTIII activity, Northern blot using a nucleic acid an acid probe specific for a gene encoding a fusion protein having GnTIII activity or a nucleic acid probe specific for a nucleic acid encoding a portion of a protein — a label fused to a polypeptide ohm having GnTIII activity, or measuring GnTIII activity. Alternatively, lectin, which binds to the biosynthetic derivatives of GnTIII, for example lecithin E4-PHA, can be used. Alternatively, a functional assay can be used to measure Fc receptor binding or enhanced effector function mediated by antibodies produced by cells that have been modified with a nucleic acid encoding a polypeptide with GnTIII activity. As another alternative, the nucleic acid may be operably linked to a reporter gene (reporter gene); expression levels of a fusion polypeptide having beta-1,4-N-acetylglucosaminyl transferase III (GnTIII) activity and containing a heterologous Golgi complex heterologous polypeptide responsible for the localization in the Golgi complex is determined by measuring the signal associated with the expression level of the reporter gene. A reporter gene can be transcribed together with nucleic acid (s) encoding a fusion protein to form a single mRNA molecule; their respective coding sequences can be linked either by an internal ribosome landing site (IRES) or by an encapsulated translation enhancer (CITE). The reporter gene can be translated together with at least one nucleic acid molecule encoding a fusion polypeptide having beta-1,4-N-acetylglucosaminyl transferase III (GnTIII) activity and containing the domain of a heterologous resident polypeptide in the Golgi complex Golgi, with the formation of a single polypeptide chain. Nucleic acids encoding the fusion polypeptides of the present invention can be operably linked to a reporter gene and placed under the control of a single promoter, and so that the nucleic acid encoding the fusion polypeptide and the reporter gene are transcribed into an RNA molecule, which by alternative splicing turns into individual molecules of messenger RNA (mRNA); one of the resulting mRNA molecules is then translated into a reporter protein, and the other is translated into a fusion polypeptide.

If several different nucleic acids are expressed that encode a fusion polypeptide having beta-1,4-N-acetylglucosaminyl transferase III (GnTIII) activity and containing a domain of a heterologous Golgi complex heterologous polypeptide responsible for localization in the Golgi complex, they can be organized as follows so that as a result of transcription one or more mRNA molecules are formed. If they are transcribed as a single mRNA molecule, the corresponding coding sequences can be connected either by an internal ribosome landing site (IRES), or by a cap-independent translation enhancer (CITE). Their transcription can be initiated by a common promoter and lead to the formation of an RNA molecule, which by alternative splicing is converted into several separate molecules of matrix RNA (mRNA), each of which is then translated into its corresponding fusion protein encoded by it.

In other embodiments, the present invention provides host cell expression systems for the production of therapeutic antibodies having enhanced binding affinity of an Fc receptor, in particular activating Fc receptors, and enhanced effector function, including antibody-dependent cellular cytotoxicity. Typically, host-cell expression systems are modified and / or selected to express nucleic acids encoding an antibody whose altered glycoforms are desired to be obtained simultaneously with at least one nucleic acid encoding a fusion polypeptide having beta-1,4-N activity -acetylglucosaminyl transferase III (GnTIII) and containing the domain of a heterologous Golgi complex resident polypeptide responsible for localization in the Golgi complex. In one embodiment, the host cell system is transfected with at least one nucleic acid encoding such a fusion polypeptide. Typically, transfected cells are selected (selected) to identify and isolate clones that stably express the fusion protein of the invention.

A cultured cell line of any type can be used as the basis for constructing cell lines according to the present invention. In a preferred embodiment, yeast cells, insect cells or plant cells can be used. Typically, such cell lines are modified to also contain at least one transfected nucleic acid encoding an entire antibody molecule, an antibody fragment containing an immunoglobulin Fc region, or a fusion protein that includes an Fc- equivalent region areas of immunoglobulin. Usually such cell

the lines for producing antibodies come from clones producing and secreting antibodies with high specific productivity in the range between 20 and 120 pg / (cell-day). In an alternative embodiment, a hybridoma cell line expressing a single target antibody is used as the basis for obtaining the modified host cells of the present invention.

In one embodiment, nucleic acids encoding an antibody, antibody fragment, or Fc fusion polypeptide are cloned into antibody expression vectors, then introduced by transfection into host cells, and cell clones with high and stable specific antibody production productivity are searched and selected. Such selected clones are then transfected with expression vectors of glycoprotein-modifying glycotransferases containing nucleic acids encoding, for example, (a) a fusion polypeptide with beta-1,4-N-acetylglucosaminyl transferase III (GnTII) activity, or (b) a fusion polypeptide with beta activity -1,4-galactosyltransferase (GalT), or (c) a fusion polypeptide with alpha-mannosidase II activity (ManII) of the Golgi complex, or (d) a fusion polypeptide with GnTIII activity as well as a polypeptide with ManII activity, or (e) a fusion polypeptide with GalT and poly activity eptid with ManII activity. The clones are then subjected to selection (by antibiotics) and among them colonies of cells are selected that are characterized by stable expression of genes encoding antibodies, and the expression levels should provide high specific productivity of antibody production, as well as colonies by stable expression of genes of glycoprotein-modifying glycotransferases, and expression levels should provide modification glycation pattern of the Fc region, including an increase in the proportion of non-fucosylated oligosaccharides (which may be branched or branched, hybrid or complex) associated with an increase in binding affinity Fc-receptor, in particular, with an increase in the affinity of Fc-FcγRIII binding and increased effector functions mediated by Fc-receptor, including but not limited to, Fc-dependent cellular cytotoxicity. Selection and selection methods are described below.

In another embodiment, the two transfections described above, namely, transfection with antibody expression vectors and transfection with expression vectors of glycoprotein-modifying glycotransferases, are carried out in reverse order, i.e. the host cell is first transfected with expression vectors of glycoprotein-modifying glycotransferases, and then with antibody expression vectors. With this approach, among the clones obtained after the first transfection, clones can be identified that give an adequate stable level of expression of glycosyltransferase genes. Identification of such clones can be carried out by any of the methods described below, or, alternatively, by temporarily transfecting the replicates of such clones with an antibody expression vector and then using the screening methods described below to identify clones with a stable expression level of glycosyltransferase genes, the expression levels must provide modification Fc region glycosylation pattern and increased binding affinity of the Fc receptor, including Fc-FcγRIII receptors, as well as an increase in Fc-mediated ffektornyh functions, including, but not limited to, Fc-dependent cellular cytotoxicity. Selection and selection methods are described below.

In a further embodiment, the genes encoding the antibody and the glycosyltransferase genes are transfected together as part of a general transfection procedure in a single expression vector or in separate vectors.

Typically, at least one nucleic acid in the host cell system encodes a fusion polypeptide having beta-1,4-N-acetylglucosaminyl transferase III (GnTIII) activity or, alternatively, beta-1,4-galactosyl transferase activity, and containing the domain of a heterologous resident Golgi complex polypeptide responsible for localization in the Golgi complex. Alternatively, the nucleic acid encodes a polypeptide with alpha-mannosidase II Golgi complex activity.

One or more nucleic acids encoding a polypeptide of the invention may be expressed under the control of a constitutive promoter or, alternatively, a regulated expression system. Suitable regulated expression systems include, without limitation: a tetracycline-regulated expression system, an ecdysone-induced expression system, an expression system containing a lac promoter, a glucocorticoid-induced expression system, a temperature-induced promoter system, and a metallothionine metal-induced expression system. If the host-cell system contains several different nucleic acid molecules encoding fusion proteins with beta-1,4-N-acetylglucosaminyl transferase III (GnTIII) activity and containing the domain of a heterologous resident polypeptide responsible for the localization in the Golgi complex Golgi complex, some of them can be expressed under the control of the constitutive promoter, while others are expressed under the control of a regulated promoter. By maximum expression level is meant the highest possible level of stable expression of a fusion protein that does not adversely affect cell growth rate. The maximum level of expression can be determined during routine experiments. Expression levels are determined by methods well known in the art, including Western blot using, for example, an antibody specific for a protein having GnTIII activity, or an antibody specific for a label segment of a protein fused to a polypeptide having GnTIII activity, Northern blot with using a nucleic acid probe specific, for example, to a gene encoding a fusion protein having GnTIII activity or a nucleic acid probe specific to a gene encoding a protein tag fused to a polypeptide, they GnTIII activity, or measurement of GnTIII activity. Alternatively, lectin can be used, which binds to the biosynthetic derivatives of GnTIII, for example, lecithin E ₄ -PHA. Alternatively, a functional assay can be used to measure increased Fc receptor binding or increased effector function mediated by antibodies produced by nucleic acid modified cells encoding a polypeptide with GnTIII activity. As another alternative, the nucleic acid may be operably linked to a reporter gene (reporter gene); fusion polypeptide expression levels are determined by measuring the signal associated with the expression level of the reporter gene. A reporter gene can be transcribed together with nucleic acid (s) encoding a fusion protein to form a single mRNA molecule; their respective coding sequences can be connected either through a sequence encoding an internal ribosome landing site (IRES), or a cap independent translation enhancer (CITE). The reporter gene can be translated together with at least one nucleic acid molecule encoding a fusion polypeptide having beta-1,4-N-acetylglucosaminyl transferase III (GnTIII) activity and containing the domain of a heterologous resident polypeptide in the Golgi complex Golgi, with the formation of a single polypeptide chain. The nucleic acid encoding the fusion polypeptide can be functionally linked to the reporter gene and placed under the control of a single (common) promoter, and so that the nucleic acid encoding the fusion polypeptide and the reporter gene are transcribed into an RNA molecule that is converted by alternative splicing to two separate molecules of messenger RNA (mRNA); one of the resulting mRNA molecules is then translated into a reporter protein, and the other is translated into the specified fusion polypeptide.

If several different nucleic acids are expressed that encode a fusion polypeptide having beta-1,4-N-acetylglucosaminyl transferase III (GnTIII) activity and containing the domain of a heterologous Golgi complex resident polypeptide responsible for localization in the Golgi complex, they can be organized in such a way so that as a result of their transcription one or more mRNA molecules are obtained. If they are transcribed to form a single mRNA molecule, the corresponding coding sequences can be linked either by an internal ribosome landing site (IRES) or by an enhancer of cap-independent translation (CGE). Their transcription can be initiated by a common promoter and lead to the formation of an RNA molecule, which, by alternative splicing, turns into several separate matrix RNA molecules (mRNAs), each of which is then translated into the corresponding encoded fusion protein.

i. Expression systems

To construct expression vectors containing the coding sequences of the target proteins and coding sequences of polypeptides with beta-1,4-N-acetylglucosaminyl transferase III (GnTIII) activity and containing the domain of a heterologous Golgi complex heterologous polypeptide responsible for localization in the Golgi complex, Together with signals controlling transcription and translation, methods well known to those skilled in the art can be used. These methods include in vitro recombinant DNA technologies, synthetic in vitro technologies, and in vivo recombination / genetic recombination. See, for example, the technologies described in Maniatis et al, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates & Wiley Interscience, New York (1989). A variety of host-expression vector systems can be used to express the coding sequences of the fusion proteins of the present invention. It is preferable to use mammalian cells transfected with recombinant expression vectors in the form of plasmid DNA or cosmid DNA containing the coding sequence of the target protein and the coding sequence of the fusion polypeptide as host-cell systems. Most preferably, yeast, insect or plant cells are used as host cell systems. Some examples of expression systems and selection methods are described in the following sources and those not referenced therein: Borth et al., Biotechnol. Bioen 71 (4): 266-73 (2000-2001), in Werner et al., Arzneimitteforschung / Drug Res. 48 (8): 870-80 (1998), Andersenand, Krummen, Curr. Op. Biotechnol. 13: 117-123 (2002), Chadd, Chamow, Curr. Op. Biotechnol. 12: 188-194 (2001), and Giddings, Curr. Op. Biotechnol. 12: 450-454 (2001). In alternative embodiments, other host-cell systems may be contemplated, including yeast cells transformed with recombinant yeast expression vectors containing the coding sequence of the target protein and the coding sequence of the fusion polypeptide, insect cells infected with recombinant viral expression vectors (eg, baculovirus) containing the coding the sequence of the target protein and the coding sequence of the fusion polypeptide, plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus (BCC, CaMV), tobacco mosaic virus (TMV)) or transformed with plasmid expression vectors (e.g., Ti plasmid) containing the coding sequence of the target protein and the coding sequence of the polypeptide fusions of the present invention, or animal cell systems infected with recombinant viral expression vectors (e.g., adenovirus, vaccinia virus), including cell lines modified with such at once to contain multiple copies of the DNA encoding the target protein and the coding sequence of the fusion polypeptide according to this invention (either stably amplified (CHO / dhfr) or unstably amplified in double-minute chromosomes (for example, in murine cells).

For the methods of the present invention, stable expression is generally preferable to transient expression because it usually gives more reproducible results and is also more suitable for large-scale production, i.e. for producing glyco-modified antibodies according to the present invention in cell lines for large-scale production. Instead of expression vector vectors containing viral origin of replication, host cells can be transformed with appropriate coding nucleic acids controlled by appropriate expression control elements (e.g., promoter, enhancer, transcription terminator sequences, polyadenylation sites, etc.), and a selective marker. After the introduction of foreign DNA, the modified cells can be left to grow for 1-2 days in an enriched medium, and then transferred to a selective medium. The selectable marker in the recombinant plasmid provides resistance to selection and allows you to select cells that stably integrate such a plasmid into their chromosomes and grow with the formation of colonies, which, in turn, can be cloned and developed into a cell line.

A variety of selection systems may be used, including, but not limited to, herpes simplex virus thymidine kinase genes (Wigler et al., Cell 11: 223 (1977)), hypoxanthine-guanine phosphoribosyl transferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48: 2026 (1962)) and adenine phosphoribosyl transferase (Lowy et al., Cell 22: 817 (1980)), which can be used in tk ^- , hgrpt ^- or aprt ^- cells, respectively. Resistance to antimetabolites can also be the basis for dhfr gene selection, which provides the cell with resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA 77: 3567 (1989); O'Hare et al., Proc. Natl Acad. Sci. USA78: 1527 (1981)), according to the gpt gene, which provides resistance to mycophenolic acid to the cell (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78: 2072 (1981)), according to the neo gene, which provides the cell with resistance to aminoglycoside G-418 (Colberre-Garapin et al, J. Mol. Biol. 150: 1 (1981)), and the hygro gene, which provides the cell with hygromycin resistance (Santerre et al., Gene30: 147 (1984). Recently, additional interesting selective genes, namely: trpB, which allows the cell to use indole instead of tryptophan, hisD, which allows the cell to use histinol instead of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. USA 85: 8047 (1988)), glutamine- synthase system and ODC (ornithine decarboxylase, ornithine decarboxylase) which provides the cell with resistance to the ornithine decarboxylase inhibitor, 2-difluoromethyl-ornithine - DFMO (McConlogue, in: Current Comniunications in Molecular Biology, Cold Spring Harbor Laboratory ed. (1987)).

ii. Identification of transfectants or transformants that express a protein with a modified glycosylation pattern.

Host cells that contain a coding sequence and which express a biologically active gene product can be identified using at least four of the following general approaches: (a) DNA-DNA or DNA-RNA hybridization, (b) the presence or absence of marker functions »Of the gene, (c) assessment of the level of transcription by expression of the corresponding mRNA transcripts in the host cell, (d) detection of the gene product by immunoassay or by the biological activity of this product.

In the first approach, the presence of the coding sequence of the target protein and the coding sequence of the fusion polypeptide of the present invention introduced into the expression vector can be detected by DNA-DNA or DNA-RNA hybridization with probes containing nucleotide sequences homologous to the corresponding coding sequences, or part or all of them derivatives.

In the second approach, the recombinant vector / host expression system can be identified and selected based on the presence or absence of certain functions of the marker gene (for example, thymidine kinase activity, antibiotic resistance, methotrexate resistance, transformation phenotype, Taurus inclusion formation in the case of baculovirus etc.). For example, if the coding sequence of the target protein and the coding sequence of the fusion polypeptide according to this invention is inserted inside the marker gene sequence of a vector, recombinants containing the corresponding coding sequence can be identified by the lack of marker gene function. Alternatively, the marker gene can be placed in tandem with the coding sequences under the control of the common or different promoters used to control the expression of the coding sequences. Expression of the marker in response to induction or selection indicates the expression of the coding sequence for the target protein and the coding sequence for the fusion polypeptide. In a third approach, the transcription activity of the coding region of the target protein region and the coding sequence of the fusion polypeptide according to this invention can be evaluated using hybridization assays. For example, it is possible to isolate RNA and analyze it by Northern blot using a probe homologous to the coding region of the target protein and the coding sequence of the fusion polypeptide according to this invention, or certain parts thereof. Alternatively, the total DNA and RNA of the host cell can be isolated and analyzed using such probes.

In the fourth approach, the expression of the protein products of the target protein and the coding sequence of a fusion polypeptide having beta-1,4-N-acetylglucosaminyl transferase III (GnTIII) activity and containing the domain of any heterologous Golgi complex resident polypeptide responsible for localization in the Golgi complex can be evaluated immunological methods, for example, using the Western blot method, immunoassay modifications, such as radioimmunoprecipitation, solid-phase immunoassay, etc. However, a crucial success test The expression system includes the determination of biologically active gene products.

b. Obtaining and using proteins and protein fragments with altered glycosylation patterns.

i. Obtaining and using antibodies with enhanced effector function, including antibody-dependent cellular cytotoxicity.

In preferred embodiments, the present invention provides glycoforms of antibodies and antibody fragments having increased Fc receptor binding affinity and / or effector function, including antibody-dependent cellular cytotoxicity.

Recent clinical trials of unconjugated monoclonal antibodies (mAbs) for the treatment of certain cancers have given encouraging results. Dillman, Cancer Biother. & Radiopharm. 12: 223-25 (1997), Deo et al, Immunology Today 18: 127 (1997). Chimeric unconjugated IgG1 has been approved for low-grade or follicular B-cell lymphoma other than Hodgkin's lymphoma (Dillman CancerBiother. & Radiopharm. 12: 223-25 (1997)), while other unconjugated MKAT, humanized IgG1 directed to solid tumors , also showed promising results in phase III clinical trials. Deo et al, Immunology Today 18: 127 (1997). The antigens of these two MKAT are actively expressed in the cells of the corresponding tumors and antibodies mediate the effective destruction of the tumor by effector cells in vitro and in vivo. In contrast to these, many other non-conjugated MKATs with high tumor specificity are not able to trigger effector functions strong enough to be clinically useful. Frost et al, Cancer 80: 317-33 (1997); Surfus et al., J. Immmother. 19: 184-91 (1996). For some of these weaker MKAT, adjunctive cytokine therapy is currently under investigation. The addition of cytokines can stimulate antibody-dependent cellular cytotoxicity (ADCC) by increasing activity and increasing the number of circulating lymphocytes. Frost et al., Cancer 80: 317-33 (1997); Surfus et al., J. Immunother. 19: 184-91 (1996). ADCC, the lytic attack of antibodies on target cells, is triggered by the binding of leukocyte receptors to the constant region (Pc) of antibodies. Deo et al., Imunology Today 18: 127 (1997). Another, but complementary to the previous, approach to increasing the activity of ADCC of unconjugated IgG1 is to modify the Fc region of antibodies. Protein modification studies have shown that FcγR receptors interact with the CH2 domain of the lower IgG loop. Lund et al., J. Immunol. 157: 4963-69 (1996). However, the binding of FcγR also requires the presence of covalently attached oligosaccharides

to conservative Asn297 in the CH2 region (Lund et al., J. Immunol 157: 4963 - 69 (1996); Wright and Morrison, Trends Biotech. 15: 26-31 (1997)), which suggests that either oligosaccharide and the polypeptide is directly involved in the formation of the interaction site, or that the oligosaccharide is necessary to maintain the active conformation of the CH2 polypeptide. Modification of the structure of oligosaccharides can, therefore, be investigated as a possible means to increase the affinity of the interaction.

The IgG molecule carries two N-linked oligosaccharides in the Fc region, one on each heavy chain. Like any glycoprotein, an antibody is produced as a set of glycoforms that have the same polypeptide backbone but different oligosaccharides attached to glycosylation sites. Oligosaccharides, usually found in the Fc region of serum IgG, belong to the complex “biaxial” type (Wormald et al., Biochemistry 36: 130-38 (1997), with low levels of sialic acid at the ends and N-acetylglucosamine at the branching site (branching, GlcNAc), as well as with a variable degree of terminal galactosylation and fucosylation of the core (base fragment). Some studies suggest that the minimum carbohydrate structure required for FcγR binding is limited to oligosaccharide core. Lund et al., J. Immunol. 157: 4963- 69 (1996)

Cell lines derived from mouse or hamster cells and used to produce unconjugated MKATs in industry and for scientific purposes usually attach oligosaccharide determinants to Fc sites. However, the immunoglobulins expressed in these cell lines lack the “branching” GlcNAc found in small amounts in plasma immunoglobulins. Lifely et al., Glycobiology 318: 813-22 (1995). Recent observations have shown that myeloma-produced humanized IgG1 (CAMPATH-1H), in contrast, carry branching GlcNAc in some of their glycoforms. Lifely et al., Glycobiology 318: 813-22 (1995). Antibodies obtained in rat cells reached the maximum in vitro activity of ADCC close to the maximum activity of CAMPATH-1H antibodies produced in standard cell lines, but at significantly lower antibody concentrations. A high level of CAMPATH antigen is usually present in lymphoma cells, and this chimeric MKAT has a high ADCC activity in the absence of a “branching” GlcNAc. Lifely et al., Glycobiology 318: 813-22 (1995). When N-glycosylation, branching GlcNAc adds the enzyme beta-1,4-acetylglucosaminyl transferase III (GnT III). Schachter, Biochem. Cell Biol. 64: 163-81 (1986).

In earlier studies, a single antibody producing CHO cell line was used that was previously modified to express, under external regulation, different levels of the cloned GnT III enzyme gene (Umana, P., et al., Nature Biotechnol. 17: 176-180 (1999 )). This approach for the first time established a strong link between the expression of GnT III and the activity of ADCC modified antibodies.

Other antibodies of the invention having enhanced Fc receptor binding affinity and enhanced effector function include, without limitation: a monoclonal antibody (chCE7) against human neuroblastoma obtained by the methods of the invention, a chimeric antibody (chG250) against human kidney carcinoma cells obtained by the methods of the invention, humanized anti-HER2 monoclonal antibody (e.g., Trastuzumab (HERCEPTIN)) obtained by the methods of the invention, chimeric monoclonal antibody (ENG-1) against carcinoma cells colon, lung, and breast nomes obtained by the methods of the present invention, humanized monoclonal antibody (3622W94) against the human antigen 17-1A, obtained by the methods of the present invention, humanized antibody (A33) against a human colon and rectum tumor, obtained by the methods of the present invention, antibody (R24) against human melanoma directed to GC3 ganglioside obtained by the methods of the present invention, as well as a chimeric monoclonal antibody (SF-25) against squamous cell carcinoma of the lung, obtained by the methods of the invention, a monoclonal antibody (BEC2, InClone Systems, Merck KgaA) against small cell lung carcinoma obtained by the methods of the invention, a monoclonal antibody (Bexxar, (tositumomab, Coulter Pharmaceuticals), Oncolym (Techniclone, Alpha Therapeutics)) against non-Hodgkin lymphoma obtained by the methods of this invention, a monoclonal antibody (C225, ImClone Systems) against squamous cell carcinoma of the head and neck of a person, prepared by the methods of the present invention, a monoclonal antibody (Panorex (edrecolomab), Centocor, Glaxo Wellcome) against colorectal carcinoma and p intestinal pit prepared by the methods of the invention, monoclonal antibody (Theragyn, Antisoma) against human ovarian carcinoma obtained by the methods of the invention, monoclonal antibody (SmartM195, Protein Design Labs, Kanebo) against acute myelogenous leukemic carcinoma obtained by the methods of the invention, monoclonal antibody ( Cotara, Techniclone, Cambridge Antibody Technology) against human malignant glioma obtained by the methods of this invention, a monoclonal antibody (IDEC-Y2B8, IDEC Pharmaceuticals) against excellent human B-cell lymphoma t Hodgkin’s lymphoma obtained by the methods of the invention, monoclonal antibody (CEA-Cide, Inimunomedics) against human solid tumors obtained by the methods of the invention, monoclonal antibody (Iodme131-MN-14, Immunomedics) against colon and rectal carcinoma obtained by the methods of the invention a monoclonal antibody (MDX-210, Medarex, Novartis) against carcinoma of the ovary, kidney, breast and prostate obtained by the methods of this invention, a monoclonal antibody (TTMA, Pharmacie & Upjohn) against carcinoma of the colon and rectum, and t also pancreas obtained by the methods of the present invention, monoclonal antibody (MDX-220, Medarex) against TAG-72 expressing human carcinoma obtained by the methods of the invention, monoclonal antibody (MDX-447) against human EGF-expressing carcinoma obtained by the methods of the present invention, monoclonal an antibody (Genentech) against VEGF (vascular endothelial growth factor), obtained by the methods of this invention, a monoclonal antibody (BrevaRex, AltaRex) against malignant melanoma and carcinoma of the breast, lung, redstatelnoy gland and human pancreas obtained by methods of the present invention, as well as a monoclonal antibody (Monoclonal Antibody Conjugate, Immune) against human acute myelogenous leukemia monoclonal antibody. Additionally, the invention provides an antibody fragment and fusion proteins containing a region equivalent to the immunoglobulin Fc region.

ii. The preparation and use of fusion proteins containing a region equivalent to the immunoglobulin Fc region, which activates Fc-mediated cytotoxicity.

As discussed above, the present invention relates to a method for increasing the binding affinity of an Fc receptor and / or the effector function of therapeutic antibodies. This is achieved by modifying the glycosylation pattern of the Fc region of such antibodies, in particular by modifying antibody producing cells to produce a polypeptide, for example, with GnTIII activity, or with GalT activity, or with ManII activity that modifies oligosaccharides attached to the Fc region of such antibodies. This strategy can be used to increase Fc-mediated cellular cytotoxicity against unwanted cells, mediated by any molecule carrying a region equivalent to the immunoglobulin Fc region, and not only therapeutic antibodies, since changes caused by glycosylation modification affect only the Fc region and, therefore, its interactions with an Fc receptor on the surface of an effector cell involved in the ADCC mechanism. Fc-containing molecules for which the methods disclosed herein are applicable include without limitation: (a) soluble fusion proteins derived from the targeting domain of a protein fused to the N-terminus of the Fc region (Chamov & Ashkenazi, Trends Biotech. 14: 52 (1996 )), and (b) membrane-bound fusion proteins obtained from are responsible for the localization in the plasma membrane of a type II transmembrane domain connected to the N-terminus of the Fc region of Stabila, P. F., Nature Biotech. 16: 1357 (1998)).

For soluble fusion proteins (a), the targeting domain directs the binding of the fusion protein to unwanted cells, such as cancer cells, i.e. similar to the binding of therapeutic antibodies. The use of the disclosed methods for enhancing effector function, including the activity of Fc-mediated cell cytotoxicity mediated by these molecules, would therefore be identical to the method used for therapeutic antibodies.

In the case of membrane-bound fusion proteins (b), unwanted cells in the body must express a gene encoding the fusion polypeptide. This can be achieved either through gene therapy approaches, i.e. by transfecting cells in vivo with a plasmid or viral vector that directs the expression of a gene encoding a fusion protein in unwanted cells, or by implanting cells genetically modified to express the fusion protein on its surface. These latter cells can be implanted into the body inside a polymer capsule (encapsulated cell therapy), in which they cannot be destroyed by the mechanism of Fc-mediated cell cytotoxicity. However, if, in the event of capsule disruption, the presence of released cells is undesirable, they can be eliminated by the mechanism of Fc-mediated cell cytotoxicity. Stabila et al., Nature Biotech. 16: 1357 (1998). In this case, the methods disclosed herein can be applied either by introducing into the vector for gene therapy an additional expression cassette that prescribes adequate or maximum expression levels of the fusion polypeptide according to the bottom invention, or by modifying the cells for implantation in order to express adequate or maximum levels of the fusion polypeptide in them according to this invention.

Therapeutic applications of antibodies, antibody fragments, and fusion polypeptides prepared according to the methods of the present invention.

Antibodies (i.e., antibodies, antibody fragments, and fusion proteins containing a region equivalent to the immunoglobulin Fc region) of the present invention can be used alone for the detection and lysis of tumor cells in vivo. It is also possible to use antibodies conjugated with suitable therapeutic agents to treat human carcinoma. For example, it is possible to use antibodies in combination with standard or conventional methods of treatment, such as chemotherapy, radiation therapy, or conjugation of antibodies with a therapeutic drug or toxin, as well as with lymphokine or tumor-inhibiting growth factor, to deliver the therapeutic agent to the site of carcinoma localization .

Conjugation techniques for such trapezoidal agents with antibodies are well known (see, for example, Amon et al., “Monoclonal Antibodies for Immunotargeting of Drugs in Cancer Therapy", in Monoclonal Antibodies and Cancer Therapy, Reisfeld et al. (Eds.), Pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", in Controlled Drug Delivery (2e ed.), Robinson et al. (Eds.), Pp. 623- 53 (Marcel Dekker, Inc. 1987); Thorpe, "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review", in Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al. (Eds.), Pp. 475- 506 (1985); and Thorpe et al, "The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates", Immunol Rev. 62: 119-58 (1982)].

Alternatively, glyco-modified antibodies can be associated with high-energy substances, for example, <131> I, which, when localized in a tumor, leads to lysis of cells at a distance of several cell diameters [See, for example, Order, "Analysis, Results, and Future Prospective of The Therapeutic Use of Radiolabeled Antibody "Cancer Therapy", in Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985)]. According to another embodiment, the antibodies of the present invention can be conjugated to a second antibody to produce an antibody heteroconjugate for treating tumors O cells as described in U.S. Patent Segal, U.S. Patent №4,676,980.

Other therapeutic applications of the antibodies of this invention include conjugation or binding, for example, using recombinant DNA technologies, to enzymes capable of converting a prodrug into a cytotoxic drug, and the use of such an antibody-enzyme conjugate in combination with a prodrug to convert a prodrug to an itotoxic drug into tumor localization site. [See, for example, Senter et al., "Anti-Tumor Effects of Antibody-alkaline

Phosphatase, "Proc. Natl. Acad. Sci. USA 85: 4842-46 (1988);" Enhancement of the in vitro and in vivo Antitumor Activites of Phosphorylated Mitocycin C and Etoposide Derivatives by Monoclonal. Antibody-Alkaline Phosphatase Conjugates ", Cancer Research 49: 5789-5792 (1989); and Senter," Activation of Prodrugs by Antibody-Enzyme Conjugates: A New Approach to Cancer Therapy, "FASEB J.4: 188-193 (1990) ].

Another use of the therapeutic antibodies of this invention includes the use, either in the presence of complement, or as part of an antibody-drug or antibody-toxin conjugate, to remove tumor cells from the bone marrow of cancer patients. According to this approach, it is possible to clean the bone marrow ex vivo by treatment with an antibody and then return to the patient's body by infusion. See, for example, Ramsay et al., "Bone Marrow Purging Using Monoclonal Antibodies", J. Clin. Immunol., 8 (2): 81-88 (1988)

Further, chimeric antibodies, recombinant immunotoxins and other recombinant constructs of the present invention having the specificity of the antigen binding region of the desired monoclonal antibody can be used in therapy. For example, the single chain immunotoxins of the invention can be used to treat human carcinoma in vivo. Similarly, a fusion protein containing at least one antigen-binding region of an antibody of the invention is attached to at least one functionally active portion of a second protein having antitumor activity, for example, lymphokine or oncostatin, can be used to treat human carcinomas in vivo. Further, recombinant techniques known in the art can be used to construct bispecific antibodies, where one of the binding specificities of the antibody is directed to the tumor-associated antigen, and the second binding specificity of the antibody is the same as the specificity of the molecule other than the tumor-associated antigen.

The present invention provides a method for the selective lysis of tumor cells expressing an antigen that specifically binds to a monoclonal antibody of the invention or its functional equivalent. This method involves the reaction of a glyco-modified antibody of the present invention, or an immunoconjugate (e.g., immunotoxin) containing the glyco-modified antibody of the present invention, with said tumor cells. These tumor cells may be carcinoma cells. Additionally, this invention provides a method of treating carcinomas (eg, human carcinomas) in vivo. This method comprises administering to a patient a pharmaceutically effective amount of a composition comprising at least one of the glycomodified antibodies of the present invention or immunoconjugates (eg, immunotoxin) containing at least one of the glycomodified antibodies of the present invention.

In a further aspect, the invention provides an improved method of treating autoimmune diseases caused in whole or in part by pathogenic antibodies, based on reducing the number of B cells and comprising administering a therapeutically effective amount of an immunologically active antibody to a person in need of treatment. The improvement consists in the appointment of a therapeutically effective amount of an antibody having an increased ADCC and prepared according to the methods of the present invention. In a preferred embodiment, the antibody is an anti-CD20 antibody. Examples of autoimmune diseases include, but are not limited to, immuno-mediated thrombocytopenia (such as acute idiopathic thrombocytopenic purpura and chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham chorea, lupus nephritis, rheumatic fever, nephritis erythematous nephritis, polygland nephritis, polygland nephritis, polygland nephritis, polygland nephritis, polygland nephritis, polygland fibrinosis, polygland nephritis, polygland nephritis, polygland nephritis, polygland nephritis, polygland fibrocystic fibrosis, polygland nephritis, polygland fibrocystic fibrosis, polyphosphoric syndrome , Takayasu arteritis, Addison’s disease, erythema multiforme (exudative), polyarteritis nodosa, ankylosing spondylitis, syndrome m Goodpasture's, thromboangiitis obliterans, primary biliary cirrhosis, Hashimoto thyroiditis, thyrotoxicosis, chronic active hepatitis, polymyositis (dermtaomyositis), polychondritis, ordinary pemphigus, Wegener's granulomatosis, malignant fibroclerotomy, spinal fibrosis, amphibole pulmonary fibrosis, amyloid fibrosis, cerebrospinal fluid syndrome, amyloid fibrosis, amyloid fibrosis, amyloid fibrosis, cerebrospinal fluid syndrome, amyloid fibrosis, amyloid fibrosis, amyloid fibrosis, amyloid fibrosis, amyloid fibrosis, amyloid fibrosis, malignant nephrolophrenia, malignant nephropathy, cerebrospinal fluid, erythematosus nephropathia , bproprogressive glomerulonephritis and fibrosing alveolitis, inflammatory reactions, such as inflammatory skin diseases, including psoriasis and dermatitis, for example, atopic dermatitis), systemic scleroderma and sclerosis, reactions associated with inflammatory intestinal diseases (such as Crohn's disease and ulcerative colitis), respiratory distress syndrome (including adult respiratory distress syndrome-RDSV), dermatitis, meningitis, encephalitis, uveitis, colitis , glomerulonephritis, allergic conditions (such as eczema, asthma and other conditions accompanied by T-lymphocyte infiltration and chronic inflammatory reactions), atherosclerosis, lymphocyte adhesion deficiency, rheumatoid arthritis, systemic edge lupus erythematosus (SLE), diabetes mellitus (e.g., type I diabetes mellitus or insulin-dependent diabetes mellitus), multiple sclerosis, Raynaud's syndrome, autoimmune thyroiditis, allergic encephalomyelitis, Sørgen's syndrome, juvenile diabetes, as well as immune reactions associated with acute and delayed hyper mediated by cytokines and T-lymphocytes, and usually found in tuberculosis, sacroidosis, polymyositis, granulomatosis and vasculitis, malignant anemia (Addison's disease), diseases accompanied by di pedogenesis of lymphocytes, inflammatory diseases of the central nervous system (CNS), multiple organ damage syndrome, hemolytic anemia (including without limitation cryoglobinemia or Coombs-positive anemia), myasthenia gravis, antigen-antibody complex mediated diseases, immune glomerular damage, antiphospholipid syndrome, allergic neuritis, Graves disease, Lambert-Eaton myasthenic syndrome, pemphigus, autoimmune polyendocrinopathies, Reiter’s disease, immobility syndrome, Behcet’s disease, giga tokletochny arteritis, immune complex nephritis, IgA-nephropathy, IgM-polyneuropathies, immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia etc. In this aspect of the invention, the antibodies of the invention are used to purify normal B cells for a long period of time.

According to an embodiment of the invention, the patient may be a person, a horse, a pig, a bull (cow), a mouse, a dog, a cat or a bird. Other warm-blooded animals are also included in the scope of the invention.

The present invention also provides a method for treating patients suffering from cancer. The patient may be a human, a dog, a cat, a mouse, a rat, a rabbit, a horse, a goat, a sheep, a cow, a chicken. Cancer can be identified as breast cancer, gall bladder cancer, retinoblastoma, papillary ovarian cystadenocarcinoma, Wilm's tumor, or small cell lung cancer, and is broadly characterized as a group of cells with a tumor-associated antigen on the surface. This method involves administering to the patient a sufficient amount of the antibody directed to the tumor attached to the cytotoxic agent for lysis of the cancer cells. Typically, the connection of an antitumor antibody with a cytotoxic agent is carried out under conditions that allow the antibody thus linked to bind its target on the cell surface. An antibody directed toward a tumor by binding to its target directly or indirectly induces or promotes cell lysis, thereby contributing to the cure of the patient.

Also provided is a method of inhibiting the proliferation of mammalian tumor cells, which comprises contacting the mammalian tumor cells with the glyco-modified antibodies of the present invention in sufficient concentration or with an immunoconjugate containing the same antibodies to inhibit the proliferation of mammalian tumor cells.

Further, the present invention provides methods for inhibiting the growth of mammalian tumor cells, treating a tumor in a patient, proliferative disease in a patient. These methods include administering to the patient an effective amount of the composition of this invention.

Thus, it is obvious that the present invention includes pharmaceutical compositions, combinations and methods of treating carcinomas in humans. For example, the invention includes pharmaceutical compositions for use in the treatment of carcinomas in humans, comprising a pharmaceutically effective amount of an antibody of the present invention and a pharmaceutically acceptable carrier.

The compositions may contain an antibody or antibody fragments, either unmodified, conjugated to a therapeutic agent (e.g., drug, toxin, enzyme or second antibody), or in recombinant form (e.g., chimeric antibodies, fragments of chimeric antibodies, bispecific antibodies). The compositions may additionally contain other antibodies or conjugates for the treatment of carcinomas (for example, a "cocktail" of antibodies).

Compositions of the present invention comprising an antibody, an antibody conjugate, and an immunotoxin can be administered for administration by conventional methods, including, but not limited to, intravenously, intraperitoneally, orally, into the lymph nodes, or directly into the tumor. The preferred route of administration is intravenous.

Various dosage forms of the compositions of the present invention are possible, including, but not limited to, liquid solutions or suspensions, tablets, pills, powders, suppositories, polymer microcapsules or microvesicles, liposomes, solutions for infusion and intravenous administration.

[0193] the Compositions according to this invention also preferably contain conventional pharmacologically acceptable carriers and adjuvants known in the art, such as human serum albumin (HSA), ion-exchange substances, alumina, lecithin, buffering agents, such as phosphates, glycine , sorbic acid, potassium sorbate, and salts and electrolytes such as protamine sulfate.

The most effective route of administration and dosage regimen of the composition of this invention depends on the severity and course of the disease, the patient’s health status and response to treatment, as well as the considerations of the attending physician. Accordingly, the dosage of the composition should be titrated for each individual patient. However, the effective dose of the composition of this invention may vary from about 1 to about 2000 mg / kg.

The molecules described herein can come in a variety of dosage forms, which include, but are not limited to, liquid solutions or suspensions, tablets, pills, powders, suppositories, polymer microcapsules or microvesicles, liposomes, as well as solutions for infusion and intravenous administration. The preferred form depends on the route of administration and therapeutic application.

The most effective route of administration and dosage of molecules according to the present invention depend on the location of the tumor being treated, the severity and course of the cancer, the patient’s health status and response to the treatment, and the considerations of the attending physician. Accordingly, the dosage of the composition should be titrated for each individual patient.

Mutual ratio of dosages for animals of various sizes and species, as well as for humans in mg / kg surface area in Freireich, E. J., et al. Cancer Chemother., Rep. 50 (4): 219-244 (1966). It is possible to adjust the dosage regimen to optimize the inhibition of growth and lysis of tumor cells, for example, it is possible to divide the dose into portions for daily administration or proportionally reduce the dose depending on the situation (for example, several divided doses can be administered daily or proportionally reduced depending on the specific therapeutic situation )

It should be obvious that in order to achieve cure, the dose of the composition according to this invention, necessary for the cure, can be reduced further, while optimizing the schedule of administration.

In accordance with an embodiment of the invention, the formational carrier may be a lipid carrier. The lipid carrier may be a phospholipid. The lipid carrier may also be a detergent. The term "detergent" here means any substance that changes (usually reduces) the surface tension of a liquid.

In one example of the present invention, the detergent may be a nonionic detergent. Examples of nonionic detergents include without limitation: Polisorbate 80 (Polysorbate 80, also known as Tween 80 or polyoxyethylene sorbitan), Brij and Triton (e.g. Triton WR-1339 and Triton A-20) .

Alternatively, the detergent may be an ionic detergent. An example of an ionic detergent includes, but is not limited to, alkyltrimethylammonium bromide.

Additionally, in accordance with the present invention, the lipid carrier may be a liposome. In this application, a “liposome" is any membrane-limited vesicle that contains any molecule of this invention or a combination thereof.

The following examples provide a more detailed explanation of the invention. The following preparation methods and examples are given to enable those skilled in the art to more clearly understand and implement the present invention. The present invention, however, is not limited to the scope of the implementation methods given in the examples, which are intended to illustrate only one aspect of the invention, functionally equivalent methods fall within the scope of this invention. Indeed, due to the following description and accompanying drawings, various modifications of the invention other than those described will become apparent to those skilled in the art. Such modifications are within the scope of the appended claims.

Examples

EXAMPLE 1

Materials and methods

1. Construction of antibody expression vectors.

The expression vector of anti-CD20 antibodies pETR1502

The expression vector of the anti-CD20 antibody C2B8, pETR1502, consists of four separate independent expression cassettes (one for the light chain of the C2B8 antibody, one for the heavy chain of the C2B8 antibody, one for the neomycin resistance gene and one for the dhfr mouse gene). All genes are under the control of the myeloproliferative sarcoma virus (MPSV) promoter and contain a synthetic consensus polyadenylation signal derived from the polyadenylation signal of the rabbit beta globin gene.

The cDNAs encoding the variable regions of the heavy chain (VH) and light chain (VL) of the anti-CD20 C2B8 antibody were assembled during a one-step process of NDP ((Kobayashi, N., et al., Biotechniques 23: 500-503 (1997))) from a set of overlapping single-stranded oligonucleotides. The original sequence encoding the VH and VL regions of the C2B8 antibody was taken from International Publication Number: WO 94/11026. The collected cDNA fragments encoding VH and VL were subcloned into the pBluescriptIIKS (+) vector, resulting in the plasmids pBlue-C2B8VH and pBlue-C2B8VH, and then sequenced.

The variable chains of the C2B8 antibody were amplified from the corresponding plasmids pBlue-C2B8VH and pBlue-C2B8VH using primers that introduced the AscI restriction site at the 5'-end and the corresponding restriction sites at the junction of the variable and constant regions (BsiWI for light chain and NheI for heavy chains). IgG1 constant regions were amplified from a human leukocyte cDNA library (Quickclone, Clontech) using primers that introduced suitable restriction sites at the 5'- and 3'-ends (BsiWI and BamHI for the constant light chain, NheI and BamHI for the constant heavy chain) .

After validation of the DNA sequences and the heavy and light chains of the C2B8 antibody were combined with the MPSV promoter and polyadenylation signals. At the first stage, two different expression vectors were constructed: one (pETR1315) for the C2B8 light chain and the other (pETR13156) for the heavy chain. At the second stage, an expression cassette containing the neomycin resistance gene (neomycin resistance gene obtained from the Tn5 transposon under the control of the minimal MPSV promoter) was introduced into the expression vector pETR1315, resulting in the plasmid pETR1481. An expression cassette containing the dhfr gene under the control of the MPSV promoter was inserted (inserted) into the vector pETR1316, resulting in plasmid pETR1328. At the final stage, both expression modules (light chain C2B8 + neomycin resistance gene and heavy chain C2B8 + dhfr gene) were combined into one vector, resulting in plasmid pETR1502.

The expression vector of anti-CD20 antibodies pETR1520

The pETR1520 vector is a combination of the expression vector of anti-CD20 antibody C2B8 with the origin (point of initiation) of Epstein-Barr virus replication (oriP) to replicate and maintain an episomal vector in cells producing the Epstein-Barr virus nuclear antigen (EBNA). To construct the C2B8 antibody expression vector, pETR1520, the C2B8 antibody expression module from the pETR1416 vector was placed as a HinDIII fragment (i.e., a fragment obtained by treatment with HinDIII restriction enzyme) in pETR1507 vector containing oriP. The vector pETR1416 is similar to the vector pETR1502 with the exception that in this plasmid, the neomycin resistance gene is controlled by the complete, not minimal, MPSV promoter.

Antifibrinonectin Antibody Expression Vector pETR1546

This vector is identical to pETR1520 except that the regions encoding the heavy and light variable chains of the anti-CD20 antibody C2D8 have been replaced by the corresponding coding regions of the human L19 antibody recognizing the ED-B domain of fibronectin. DNAs encoding the variable regions were synthesized by overlapping extension PCR using synthetic oligonucleotides based on the sequence of the variable regions of the L19 antibody (Pini, A. Et al. J. Biol. Chem. 273 (34): 21769-76 (1998) ) The expression vector of anti-EGFR antibodies (C225 *) pURSI28

This vector is identical to pETR1520, except that the regions encoding the heavy and light variable chains of the anti-CD20 C2B8 antibody were replaced by the corresponding coding regions of the C225 antibody, a chimeric antibody that recognizes the human epidermal growth factor receptor. DNA fragments encoding the variable regions of the C225 antibody were synthesized by overlapping extension PCR using synthetic oligonucleotides based on the sequence of the variable regions of the C225 antibody (the sequence can be found in published patent application, international publication number WO 96/40210, in Fig. 16 and Fig. 17 which shows the sequences of the heavy and light chains, respectively).

2. Construction of expression vectors of the GnTIII fusion polypeptide pETR1166. Vector for constitutive expression of GnTIII.

To construct the GnTIII expression vector, pETR1166, rat GnTIII obtained from rat kidney cDNA library (Clontech) was amplified by PCR. For convenience, the subsequent determination of GnTIII by Western blot immediately after the stop codon of the GnTIII gene in the direction “against transcription” (irzgeate) was added the C-terminal portion of the c-myc epitope (amino acid sequence: PEQKLISEEDL). After confirming the correctness of the GnTIII sequence, the gene was interfered with the control of the MPSV promoter and the rabbit beta globin polyadenylation synthetic signal was added. The final GnTIII expression vector also contained a separate selective cassette with the puromycin resistance gene also under the control of the MPSV promoter and with a synthetic rabbit beta globin polyadenylation signal.

pETR1425: Replacing the first 76 amino acids of GnTIII with the first 102 amino acids of human GnTI.

The construction of the gene for this hybrid glycosyltransferase was performed by sequential overlapping PCR reactions. In one reaction, the stem region of the GnTI was amplified using primers GAB-179 and GAB-180. During this PCR reaction, a Kozak consensus sequence and an Ascl restriction site were introduced at the 5'-end. The resulting PCR fragment contains an overlapping region with the 23np Gnlll gene starting at position 229. During the second PCR reaction using GAB-177 and GAP-178 primers, the Gnlll region between positions 229 and 380 (including 229 and 380). As a result, a fragment was obtained containing a unique BstXI site at the 3'-end and an overlapping site with a GnTI core region 22 np long at the 5'-end. Both fragments obtained by PCR were purified and used as templates in the third PCR reaction (primers: GAB-179 and GAB-178). The resulting fragments were purified, digested with restriction enzyme AcsI and ligated with the vector pETR1001 (cut by AscI and SmaI), resulting in the obtained plasmid pETR1404. After the correct insertion sequence was confirmed, the MPSV promoter element was added to the vector pETR1404 as an AscI (partial cleavage) / PmeI fragment. As a result, plasmid pETR1423 was obtained. Then, the SphI / BstXI fragment of the pETR1166 vector, the expression vector carrying the original rat GnTIII gene, was replaced by the corresponding fragment of the pETR1425 vector containing the GnTI-GnTIII fusion polypeptide gene under the control of the MPSV promoter and a cassette containing the puromycin resistance gene (for selection)

Primer Sequences:

GAB-177: GCGTGTGCCTGTGACCCCCGGGCCCCTGCTCCAGCCACTGTCCCC

GAB-178: GAAGGTTTCTCCAGCATCCTGGTACC

GAB-179: CTGAGGCGCGCCGCCACCATGCTGAAGAAGCAGTCTGCAGGGC

GAB-180: GGGGACAGTGGCTGGAGCAGGGGCGCGGGGGTCACAGGCACACGCGGC

pETR1506: Replacement of the first 76 N-terminal amino acids of GnTIII with the first 102 amino acids of human mannosidase II.

The construction of the pERT1506 vector was carried out similarly to the construction of pETR1425. The core region of human mannosidase II was amplified with primers GAB-252 and GAB-253 using the pBlue-man vector as a template. During PCR, the Fse site and the Kozak consensus sequence were introduced at the 5'-end. The resulting PCR fragment overlaps the GnTIII gene in the 23 bp region starting at position 229. During the second PCR reaction, a portion of the GnTIII gene (positions 229-460) with primers GAB-254 and GAB-255 was amplified. This PCR reaction generated a fragment containing a region of overlap with the mannosidase II gene with a length of 43 n.p. at the 5'-end and the unique StuI site at the 5'-end. Both fragments were purified and used as matrices in the third PCR with the GAB-252 and GAB-255 primers. The resulting fragment was introduced into the vector pIC19H, which gave the vector pETR1484. After the correct sequence of the insert was confirmed, a complete fusion gene was constructed by ligation of the FseI / StuI fragment of the vector pETR1484 with the Fsel / BamHI fragment of the vector pETR1166 into the vector pETR12177 (FseI / BamHI). The resulting plasmid (pETR1500) contained the ManII-GnTIII fusion gene (SEQ ID NO: 12) under the control of the MPSV promoter. To select a plasmid in a mammalian cell, a cassette containing the puromycin resistance gene was introduced (in the form of a SpeI fragment from the vector pETR1166), as a result of which the plasmid pETR1506 was obtained.

Primer Sequences:

GAB-252:

GCTAGGCCGGCCGCCACCATGAAGTTAAGCCGCCAGTTCACCGTGTTCGG

GAB-253:

GGGGACAGTGGCTGGAGCAGGGGGTGAGCCAGCACCTTGGCTGAAATTGCTTT

GTGAACTTTCGG

GAB-254:

TCCGAAAAGTTCACAAAGCAATTTCAGCCAAGGTGCTGGCTCACCCCTGCT

CCAGCCACTGTCCCC

GAB-255: ATGCCGCATAGGCCTCCGAGCAGGACCCC

pETR1519: Combination of the hybrid gene gene fusion polypeptide ManII-GnTIII with the origin of replication oriP of the Epstein-Barr virus.

Using primers GAB-261 and GAB-262, an oriP-containing fragment of 2 kb of plasmid pCEP4 (Invitrogen) was amplified. During the PCR reaction, SspI and EcoRI sites were introduced at both ends of the fragment. For sequencing, a fragment with oriP was introduced into pICI19H vector. After the sequence was confirmed, oriP was introduced as an SspI fragment into pETR 1001 vector (digested with BsmBI restriction enzyme, overlapping 5'-ends were filled using Klenov polymerase). The resulting plasmid was designated as pETR1507. The expression cassette ManII-GnTI in the form of an SphI / NruI fragment of the vector pETR1510 was introduced into the vector pETR1507, cleaved with the same endonucleases, resulting in plasmid zETR1519.

Primer Sequences:

GAB-261: GCTAAATATTGAATTCCCTTTATGTGTAACTCTTGGCTGAAGC

GAB-262: TAGCAATATTGAATTCGCAGGAAAAGGACAAGCAGCGAAAATTCACGC

pETR1537: Combination of the fusion gene gene fusion polypeptide ManII-GnTIII and the truncated gene marker of the cell surface marker CD4.

The expression vector pETR1506 was modified to further express the truncated CD4 cell surface marker gene. Briefly, the expression cassette of the hybrid gene of the fusion polypeptide ManIII-GnTIII was converted from monocistronic to bicistronic expression cassette by insertion, in the direction "in the direction of transcription" from the stop codon of the gene of the fusion protein ManIII-GnTIII, the polio virus IRES element, and the next cDNA encoding a truncated human CD4 protein (containing the human CD4 leader sequence necessary for secretion, and the transmembrane and extracellular domains located behind it).

3. Transfection of mammalian cells with expression vectors of fusion polypeptide GnTIII and antibodies.

VKN Cell Transfection

Cells in the exponential growth phase (viability of 90-95%) were seeded in the corresponding number of T75 vials at a concentration of 0.9 × 10 ⁶ cells / ml 24 hours before electroporation. An Invitrus culture medium (Cell Culture Technologies, Switzerland) supplemented with 10% fetal cattle serum was used. Before electroporation, the number of cells was counted. 8 × 10 ⁶ cells were harvested by centrifugation (5 min, 200g) and the supernatant was removed. Cells were resuspended in 800 ml of Invitrus medium and transferred to sterile electroporation cuvettes (0.4 cm gap) containing 8 μg of circular plasmid DNA, then incubated for 5 min at room temperature. Cells were electroporated in a Gene Pulser II (BioRad) electroporator under the following conditions: 400V, 960 μF, two pulses in 30 sec. interval. After electroporation, the cells were immediately transferred to a 25T vial containing 5 ml of growth medium (Invitrus / 20% vol. Fetal serum / 1.25% vol. DMSO), incubated at 37 ° C in an incubator with an atmosphere containing 5% CO ₂ . To obtain unchanged (non-glyco-modified) antibodies, cells were transfected with antibody expression vectors only. To obtain glyco-modified antibodies, the cells were co-transfected with two plasmids, one for expressing the antibody and the other for expressing the GnTIII fusion polypeptide (SEQ ID NO: 15) in a ratio of 3: 1, respectively.

HEK293-ENBA Cell Transfection

HEK293-ENBA cells in the exponential growth phase were transfected with the calcium phosphate method. Cells were grown as an adhesive monolayer culture in T vials with DMEM supplemented with 10% fetal serum, and transfected when the continuity of the layer was in the range of 50-80. For transfection of a T75 vial, 8 million cells were seeded in 14 ml of DMEM culture medium supplemented with fetal serum (final ratio 10% vol.) And 250 μg / ml neomycin 24 hours before transfection, then the cells were placed overnight at 37 ° C in an incubator with an atmosphere containing 5% CO ₂ . For each T75 vial to be transfected, a solution was prepared consisting of DNA, CaCl ₂ and water. To do this, 47 μg of total DNA of the plasmid vector, 235 μl of a 1M CaCl ₂ solution were mixed, and water was adjusted to a final volume of 469 μl. To this solution was added 469 μl of a solution containing 50 mM HEPES, 280 mM NaCl and 1.5 mM Na ₂ HPO ₄ , pH 7.05, immediately stirred for 10 seconds, and left to stand at room temperature for 20 seconds. The suspension was diluted with 12 ml of DMEM medium supplemented with 2% fetal serum and added to T75 instead of the existing medium. Cells were incubated at 37 ° C, 5% CO ₂ for approximately 17-20 hours, then the medium was replaced with 12 ml of DMEM with 10% fetal serum. To obtain unchanged (non-glyco-modified) antibodies, cells were transfected with antibody expression vectors only. To obtain glyco-modified antibodies, cells were co-transfected with two plasmids, one for expressing the antibody and the other for expressing the GnTIII fusion polypeptide at a ratio of 4: 1, respectively. On the 5th day after transfection, the supernatant was collected, centrifuged for 5 minutes at 1200 rpm, and then centrifuged for a second time (4 minutes at 4000 rpm). The supernatant was stored at 4 ° C.

Obtaining stable cell lines expressing a recombinant anti-CD20 antibody

BHK cells (BHK21-13c) were transfected with the expression vector pETRT502 of antibody C2B8, which contains the expression cassette of the neomycin resistance gene, by electroporation. First, neomycin-resistant clones were selected to obtain a set of clones that integrated the DNA of the vector pETR1502 into chromosomes. Clones were then examined for the production of recombinant antibodies using solid-phase ELISA. Briefly, 24 hours after electroporation, viable cells were counted and transfection efficiency was determined by counting fluorescent cells in parallel electroporation control with the pEYFP expression vector. Cells were diluted with Invitrus selective medium containing 10% fetal serum and 1 mg / ml neomycin. Typically, eight 96-well plates were seeded with viable transfected cells at different concentrations (1 × 10 ³ , 5 × 10 ² and 1 × 10 ² cells / well) and incubated at 37 ° C until clones could be identified. As monolayers of clone cells became almost continuous, their supernatants were analyzed by solid phase ELISA. ELISA-positive clones were transferred to large-capacity containers: first into 24-well plates, then into 6-well plates, and then into T25 vials. After 5 days of growth, the final antibody titer was determined in T25 vials using a solid-phase ELISA kit. Using electroporation and selection procedures, a clone of BHK was isolated expressing the anti-CD20 antibody C2B8, BHK-1502-28, which produced 13 μg / ml of antibody under the cultivation conditions described above.

Obtaining stable mammalian cell lines expressing a recombinant anti-CD20 antibody and GnTIII fusion polypeptide.

Clone BHK-1502-28, constitutively expressing the anti-CD20 monoclonal antibody genes and the neomycin resistance gene, was transfected with the expression vector pETR1537 by electroporation. pETR1537 is a vector for constitutive expression of the ManII-GnTIII gene and the truncated form of CD4, and the expression of the truncated form of CD4 is IRES-dependent. The vector also contains an expression cassette of the puromycin resistance gene. Puromycin-resistant clones were first selected in order to obtain a set of clones that integrated the DNA of the vector pETR1537 into their chromosomes. The clones were then examined for expression on the surface of truncated CD4 (tCD4), which serves as a marker of the expression level of the bicistronic expression unit of the ManII-GnTIII + tCD4 gene. The production of recombinant antibodies by selected clones was verified by solid phase ELISA.

Briefly, the vector DNA of pETR1537 was linearized by Xmnl restriction. At the same time, control transfection with the expression vector of the EYFP fluorescent protein was performed. Transfection efficiency was determined after 24 hours, counting the ratio of the number of cells expressing EYFP to the total number of cells. 58% of the cells expressed EYFP. Cell viability was 91%. 1 day after transfection, serial dilutions (1: 100.1: 500: 1: 1000 and 1: 5000) of cells transfected with pETR1537 or pEYFP vectors were performed and then plated in 96-well plates with selective medium (final volume 0.2 ml, composition: Invitrus, 10% fetal serum, 20 μg / ml puromycin, 1 mg / ml neomycin). Clones became visible after 2 weeks. They were propagated and examined for CD4 (tCD4) expression and antibody expression.

To study the expression level of tCD4, approximately 500,000 cells were washed with FACS buffer and incubated on ice with 5 ml P1TC-labeled (FITC, fluorescein isothiocyanate) anti-CD4 h antibodies (Becton Dickinson, Switzerland) for 10 minutes. After two washes, the cells were resuspended in 0.5 ml FACS buffer and analyzed by the "fluorescence activated cell sorting" (FACS) method (Fig. 17A-B). Thus, a clone with a good level of tCD4 expression was isolated (BHK-1502-28-11). After 5 days of growth in a T25 vial, he produced anti-CD20 antibodies with a final titer (determined by solid-phase ELISA) of approximately 17 μg / ml.

4. Obtaining and purification of unchanged and glyco-modified antibodies.

The collection of culture medium

In the case of BHK cells transfected with an antibody expression vector or cotransfected with an antibody expression vector and an expression vector of a GnTIII fusion polypeptide, culture supernatant was collected after 96 hours of culturing the transfected cells after transfection. Depending on the expected productivity for a single vector, several (10-15) electroporations were performed.

In the case of HEK293-EBNA cells transfected with an antibody expression vector or cotransfected with an antibody expression vector and an expression vector of a GnTIII fusion polypeptide, the culture medium was replaced with fresh culture medium approximately 16 hours after transfection. Fresh culture medium was then collected after an additional 120 hours of culturing transfected cells.

For a stable cell line, BHK-1502-28-11, the culture was seeded at a concentration of 500,000 cells / ml, and the supernatant was collected after 4 days of cultivation, when the cell density was 1.7 × 10 ⁶ viable cells / ml with a viability of 69%.

Antibody purification

The monoclonal antibody was purified from the culture supernatant through two successive chromatography steps. The first stage consisted of chromatography using protein A with elution in a pH gradient that effectively separates human and bovine IgG. This step was followed by a cation exchange [romatography] step to replace the sample buffer with a phosphate buffered saline (PBS).

5. Analysis of oligosaccharides

Oligosaccharides were cleaved from PVDF immobilized on the membrane (Ha based on polyvinylidene fluoride) or dissolved antibodies (released) by enzymatic means: by means of PNGaseF glycosidase cleavage.

The resulting cleavage solution containing the released oligosaccharides was either immediately prepared for analysis on a MALDI / TOF-MS mass spectrometer, or subjected to further digestion with EndoH endoglycosidase before analysis for analysis on a MALDI / TOF-MS mass spectrometer.

Method for the release of oligosaccharides for membrane-immobilized PVDF antibodies.

Wells of a 96-well PVDF membrane-coated plate (Immobilon P, Millipore, Bedford, Massachusetts) were wetted with 100 μl of methanol, and the liquid was pumped through the PVDF membrane under the vacuum created by the Multiscreen vacuum manifol filtration system (Millipore, Bedford, Massachusetts). PVDF membranes were washed three times with 300 μl of water. The wells were then washed with 50 μl of RCM buffer (8M urea, 360 mM Tris, 3.2 mM EDTA, pH 8.6). 30 to 40 μl of antibody was placed in a well containing 10 μl of RCM buffer. The liquid was suctioned through the membrane under vacuum, then the membrane was washed twice in succession with 50 μl of RCM buffer. Disulfide bridges were destroyed by adding 50 μl of dithiothreitol in RCM buffer and incubation at 37 ° C for one hour.

After the bridges were destroyed by vacuum, the dithiothreitol solution was removed from the well. The wells were washed three times with 300 μl of water, and then the cysteine residues were carbomethylated by adding 50 μl of 0.1 M iodoacetic acid in RCM buffer and incubated for 30 min at room temperature.

After carbomethylation, the cells were dried under vacuum and then washed 300 times with 300 μl of water. The PVDF membrane was then blocked to prevent adsorption of endoglycosidase by incubating 100 μl of a 1% aqueous solution of polyvinylpyrrolidone 360 at room temperature for one hour. Then the blocking reagent was removed with a soft vacuum with three subsequent washes with 300 μl of water.

N-linked oligosaccharides were released by adding 2.5 mU peptide N-glycosidase F (PNGaseF recombinant N-glycanase, recombinant N-Glykanase, GLYKO, Novato, CA) and 0.1 mU Sialidases (Sialidase, GLYKO, Novato, California) to remove charged residue of monosaccharides. The final volume is 25 μl in a solution of 20 mm NaHCO ₃ , pH 7.0. Cleavage was carried out at 37 ° C for 3 hours.

The release method for antibodies in solution

From 40 to 50 μl of antibody was mixed with 2.5mU PNGaseF glycosidase (Glyko, USA) in 2 mM Tris (pH 7.0) (final volume of 25 microliters); the mixture was incubated at 37 ° C for 3 hours.

The use of Enodoglycosidase H cleavage of PNGaseF-released oligosaccharides by glycosidase PNG to match the structure of hybrid branched oligosaccharides to peaks of neutral oligosaccharides. obtained on a MALDI / TOF-MS mass spectrometer

The glycosidase released PNGaseF was then digested with Endoglycosidase H (EndoH, EC 3.2.1.96). For endoglycosidase H cleavage, the solution obtained after PNGaseF glycosidase cleavage (antibody in solution, method described above) was added with 15 mU EndoH to obtain a final volume of 30 microliters, the mixture was incubated for 3 hours at 37 ° C. EndoH cleaves oligosaccharides between N-acetylglucosamine residues of the chitobiotic core of N-linked oligosaccharides. The enzyme breaks down only oligomannose type glycans (containing only N-acetylglucosamine and mannose) and most hybrid type glycans, whereas complex oligosaccharides of this type are not hydrolyzed.

Sample preparation for mass spectroscopy with a MALDI / TOF-MS

Enzymatic cleavage products containing the released oligosaccharides were incubated for another 3 hours at room temperature after addition of acetic acid (final concentration 150 mM), and then passed through 0.6 ml of cation exchange resin (AG50W-X8 resin, hydrogen form, granules 100-200, BioRad, Switzerland), enclosed in a Micro Bio-Spin chromatography column (BioRad, Switzerland), to remove cations and proteins. One microliter of the resulting sample was applied onto a stainless steel target plate and mixed on it with 1 μl sDHB matrix. The sDHB matrix was prepared as follows: 2 mg of 2,5-dihydrobenzoic acid and 0.1 mg of 5-methoxysalicylic acid were dissolved in 1 mg of a solution of the composition: ethanol / 10 mM aqueous solution of sodium chloride (1/1 by volume ratio). The samples were air dried, 0.2 μl of ethanol was applied, and finally allowed to crystallize in air.

MALDI / TOF-MS

To obtain mass spectra, we used a MALDI-TOF mass spectrometer, model Voyager Elite (Perspective Biosystems). The device worked in linear mode with an acceleration of 20 kV and a delay of 80 ns. To establish the correspondence of the masses of ions, external calibration with standard oligosaccharides was used. To obtain the final spectrum, the spectra obtained from 200 laser pulses of 200 pulses were combined.

6. Preparation of MNPK

Peripheral blood mononuclear cells (PBMCs) were prepared using Histopaque-1077 (Sigma Diagnostics Inc., St. Louis, M063178, USA), strictly following the manufacturer's instructions. Briefly, from volunteers who ran at full speed for 1 minute to increase the percentage of natural killer cells (NK cells), venous blood was taken with heparin syringes. Blood was diluted with an FBI that did not contain Ca and Mg in a ratio of 1: 0.75-1.3 and applied to Histopaque-1077. The gradient was centrifuged at 400 × g without interruption for 30 minutes at room temperature (CT). A fluid adjacent to the Histopaque border containing PBMCs was collected, washed with PBS (50 ml per cell from two gradients), and PBMCs were separated by centrifugation at 300 × g for 10 minutes at RT. After resuspending the pellet at the FBI, the PBMCs were counted and washed a second time by centrifugation at 200 × g for 10 minutes at RT. Then the cells were resuspended in a suitable medium for subsequent procedures.

7. Isolation of NK cells

Human NK cells were isolated from MNPC using a negative selection procedure using magnetic granules that did not bind CD16 and C056 positive cells ((MACS system, Miltenyi Biotec GmbH, 51429 Bergisch Gladbach, Germany)). MNPCs were washed once with ice-cold MACS buffer (FBI containing 2% fetal serum and 2 mM EDTA), resuspended at a concentration of 20 million cells per ml of a mixture of fetal serum and MACS buffer (1: 1), and then incubated for 10 min at 4 ° C. Then the cells were besieged and resuspended in MACS buffer containing 10% fetal serum (80 μl of buffer per 10 million cells). Then, 20 μl of Hapten-antibody solution per 10 million cells was added. Cells were incubated for 10 min at 4 ° C, constantly rotating the tubes. After two washes in the MACS buffer (in at least 10 labeling volumes of the buffer) After a single wash in the MACS buffer (in at least ten times the volume), the cells were resuspended in the MACS buffer containing 10% fetal serum at a concentration of 10 million cells per 80 μl and 20 μg of anti-hapten microbeads (i.e., microbeads coated with anti-hapten antibodies) for every 100 million cells were added. The tubes were incubated for 15 minutes at 4 ° C, constantly rotating the tubes. After one wash with MACS buffer, cells were resuspended to a concentration of up to 100 million cells in 500 μl of MACS buffer and loaded onto a LS MACD column, which was placed in a MINI-MACS magnet and equilibrated with 3 ml of MACS buffer. The column was washed three times with 3 ml of MACS buffer.

Cells were collected in the flow phase and subsequently used as NK cells. The purity, determined by the expression of CD56, was 88-95%.

8. Analysis of the ACCC

Batch or NK as effector cells were prepared as described above. The ratio of effector cells to target cells was 25: 1 or 10: 1 for MNPC and NK cells, respectively. Effector cells were prepared in AIM-V medium at a concentration suitable to place 50 μl in each well of a 96-well plate. The target cells of C2B8 antibodies were SKW6.4 cells or Namalwa B-lymphoblastoid cells grown in DMEM medium containing 10% fetal serum. Target cells were washed in PBS, counted and resuspended in AIM-V medium at a concentration of 0.3 million cells per ml in order to place 30'000 cells in 100 μl of solution in each well of the microplate. Antibodies were diluted in AGM-V medium, 50 μl of the resulting solution was added to target cells preliminarily coated onto the plate and allowed to bind for 10 min at RT. Then, effector cells were added and the plate was incubated for 4 hours at 37 ° C in a humid atmosphere containing 5% CO ₂ . Lysis of target cells was determined by measuring the release of lactate dehydrogenase (LDH) from damaged cells using the Cytotoxicity Detection kit cytotoxicity kit (Roche Diagnostics, Rothcruise, Switzerland). After four hours of incubation, the plates were centrifuged at 800 x g. 100 μl of supernatant from each well was transferred to a new, clear, flat-bottomed 96-well plate. 100 μl of the colored substrate from the kit was added to each well. The Vmax values of the color reaction were determined on a solid-phase ELISA reader (wavelength 490 nm) for at least 10 minutes using the SOFTmax PRO software (Molecular Devices, Sunnyvale, CA94089, USA). Spontaneous LDH releases were measured in wells containing only target cells and effector cells, without antibodies. Maximum release was determined in wells containing only target cells and 1% Triton X-100. The percentage of specific antigen-mediated lysis was calculated as follows: ((x-SR) / MR-SR) * 100, where x is the average Vmax for a given concentration of antibodies, SR is the average Vmax of spontaneous release, MR is the average Vmax of maximum release.

9. Binding of FcγRIIIA Receptor on NK Cells

Freshly isolated NK cells were centrifuged for 5 minutes at 200xg and etched with 0.09% (w / v) lactic acid solution (140 mM NaCl, 5 mM KCl, pH 3.9) by incubation at a concentration of 3 × 10 ⁵ cells / ml at room temperature in within 5 minutes of removal for NK-associated IgG (De Haas M., J. Immmunol. 156: 2948 (1996)).

Cells were washed twice with a mixture of PBS, 0.1% BSA, 0.01% sodium azide and the concentration was adjusted to 2 × 10 ⁶ cells / ml in a mixture of PBS, 0.1% BSA, 0.01% sodium azide. 5 × 10 ⁵ cells were incubated for 30 minutes at 4 ° C with 0, 0.1, 0.3, 1, 3, and 10 mg / ml antibody variants. The cells were then washed twice and antibody binding was determined by incubation with the fluorescein conjugate of isothiocyanin and goat IgG against human F (ab ') ₂ (1: 200) and with the conjugate of anti-human CD56 antibody and PE (phycoerythrin) (5 μl / 5 × 10 ⁵ cells) for 30 minutes at 4 ° C (Shields R. Et al. J. Biol. Chem. 277 (30): 26733-26740 (2002)). Goat IgG against human F (ab ') 2 was obtained from Jackson hnmunoReasearch, West Grove, PA, and anti-hC56-PE-BD Pharmingen conjugate, San Diego, California.

The fluorescence intensity corresponding to the bound antibody variants was determined for CD56 + cells on a FACSCalibur instrument (BD Bioscience, San Jose, California).

10. Binding of the FcgammaRIIb receptor on Raji lymphoma cells.

Raji line B lymphoma cells were washed for 20 min at 37 ° C in the FBI (concentration of 0.3 million cells / ml). Then the cells were resuspended at a concentration of 2.22 million cells / ml in a mixture of PBS, 0.1% BSA, 0.01%) NaN ₃ and 180 μl of the resulting suspension was added to each FACS tube. Unchanged L19 antibodies and glyco-modified L19 antibodies in tenfold dilutions (0, 0.1, 0.3, 1, 3, 10, 30 μg / ml) were added to Raji cells and incubated at 4 ° C for 30 minutes (final cell concentration 2 million cells / ml). After two washes, a fluorescin isothiocyanin conjugate with goat IgG against human F (ab ') ₂ conjugate (Jackson ImmunoReasearch, West Grove, PA) was added in a ratio of 1: 200 and incubated at 4 ° C for 30 minutes. After one wash, the cells were resuspended in 0.5 ml of a mixture of PBS, 0.1% BSA, 0.01% NaN _3, and the fluorescence intensity associated with the bound antibody variants was determined on a FACSCalibur instrument (BD Bioscience, San Jose, California).

35

11. Analysis of complement-dependent cell cytotoxicity

Target cells were counted, washed in PBS, resuspended in AIM-V medium (Invitrogen) at a concentration of 1 million cells / ml. 50 μl of cells were added to the wells of a flat-bottomed 96-well plate. Antibodies were diluted in AIM-V medium and 50 μl were added to the cells. Then the antibodies were left to bind to cells for 10 minutes at room temperature. Freshly thawed human serum complement (Quidel) was diluted three times with AIM-V medium and added in a volume of 50 μl to the wells. Rabbit complement (Cedarlane Laboratories) was prepared as described by the manufacturer, diluted three times with AJM-V medium and added in a volume of 50 μl to the wells. As a control, complement sources warmed up for 30 minutes at 56 ° C were used.

The assay plates were incubated for 2 hours at 37 ° C. Cell lysis was determined by the release of LDH. Briefly, the plates were centrifuged at 300 x g for 3 minutes. 50 μl of the supernatant from each well was transferred to a new 96-well plate, and 50 μl of the cytotoxicity kit of the Cytotoxity Kit (Roche) was added. During kinetic measurements on a reader for solid-phase ELISA, the value of Vmax corresponding to the concentration of LDH in the supernatant was determined. Maximum release was determined by incubating cells with 1% Triton X-100.

Results and discussion

Glyco-modified variants of the anti-CD20 chimeric IgG1 antibody (chimeric C2B8 antibody, also known as rituximab) were obtained by cotransfection of mammalian cells with antibody expression vectors and gene expression vectors encoding various polypeptides with GnTIII activity. Unchanged (non-glyco-modified) variants of the same antibodies were obtained by transfecting mammalian cells with the antibody gene expression vector only. Transfected cells were cultured for at least 3 days, then secreted recombinant antibodies were purified from the culture medium by affinity chromatography using protein A. Expression of genes encoding a polypeptide with GnTIII activity did not significantly affect cell viability, cell growth or antibody production compared to with cells that do not produce such polypeptides.

The object of analysis was the glycosylation pattern of purified antibodies. These antibodies carry N-linked oligosaccharides attached exclusively to the Asn297 residue of the human IgG1 Fc region. Oligosaccharides were separated from antibodies by enzymatic means (using PNGaseF glycosidase digestion) and then analyzed on a MALDI / TOF-MS mass spectrometer. This technique allows the share of different types of oligosaccharides in the total, initial, population of oligosaccharides associated with the Fc region, as well as to establish the correspondence of structures to different peak spectra (Umana, P. et al., NatureBiotechnol. 17: 176-180 (1999)).

In FIG. 1 shows the MALDI / TOF-MS spectra (compositions) of neutral oligosaccharides of the chimeric anti-CD20 IgG1 antibody produced by BHK cells. These cells were transfected with the antibody expression vector pETR1502. In FIG. 2 - FIG. 4 shows the corresponding spectra for the same antibody produced by BHK cells modified with nucleic acids encoding wild-type GnTIII. The spectrum in FIG. 2 is obtained by using a nucleic acid encoding wild-type GnTIII expressed from pETR1166 vector. The spectrum in FIG. 3 was obtained by using a nucleic acid encoding a fusion polypeptide containing, at the N-terminus, a GnTI localization domain fused to the C-terminal GnTIII catalytic domain. This fusion gene was expressed from pETR1425 vector. The spectrum in FIG. 4 was obtained by using a nucleic acid encoding a fusion polypeptide containing, at the N-terminus, a Golgi alpha-mannosidase II localization domain (ManII) fused to the GnTIII catalytic domain. This fusion gene was expressed from pETR1506 vector.

An unchanged antibody has a typical spectrum (composition) of oligosaccharides (Fig. 1), in which there are peaks with m / z ratios of 1485, 1648 and 1810, corresponding to bi-antenna corfucosylated complex oligosaccharides with the number of galactosidase residues 0, 1 and 2, respectively. Similar spectra show oligosaccharides of the Fc region of unmodified IgG1 class antibodies produced by other standard industrial mammalian cell lines, such as CHO and mouse myeloma cells (Lifely, M. R. et al., Glycobiology 5: 813-822 (1995)). Modification of antibodies produced by cells by expression of wild-type GnTIII leads mainly to the appearance of branched complex two-antenna oligosaccharides (Fig. 2) with peaks at m / z ratios of 1689, 1851 and 2014, which correspond to branched copies of unbranched peaks

fucosylated oligosaccharides found in an unmodified antibody. Modification of antibody-producing cells by expression of a nucleic acid encoding the GnTI-GnTIII fusion polypeptide, whose GnTIII catalytic domain is localized by the GnTI domain responsible for localization in the Golgi complex, also leads mainly to the appearance of branched complex two-antenna oligosaccharides (note the peaks with m / z 1689 and 1851 in Fig. 3) However, with respect to wild-type GnTIII, the use of the GnTI-GnTIII fusion protein leads to an increase in the proportion of branched non-fucosylated and branched hybrid structures p (cf. the ratio of peaks with m / z 1664, 1810, 1826 and 1973, the total in FIG. 2 and FIG. 3). The synthesis of branched non-fucosylated and branched hybrid structures results from competition for GnTI-modifiable substrates between the catalytic domain of recombinant GnTIII and (i) endogenous alpha-1,6-core-fucosyltransferase, (ii) alpha-mannosidase II (ManII) of the Golgi complex and ( iii) GnTIIII; since the oligosaccharide modified during the GnTIII-catalyzed reaction by the addition of N-acetylglucosamine at the branching site (“branching GlcNAc”) cannot then be modified by the other three enzymes. Therefore, the blocking effect of ManII adding branching GlcNAc also effectively blocks GnTII, since GnTII acts in the direction of the synthesis pathway of N-linked oligosaccharides “lower” than ManII. Peaks with m / z 1664 and 1826 are not fucosylated oligosaccharides, but with m / z 1810 and 1973 are fucosylated. To confirm that the increase in these peaks is due to an increase in the proportion of branched nefucosylated and branched hybrid oligosaccharides associated with the Fc region (Fig. 8A), EndoH glycosidase cleavage was used, which is able to "distinguish" hybrid and complex oligosaccharides (see below).

Modification of antibody producing cells by expression of a nucleic acid encoding a ManII-GnTIII fusion polypeptide (SEQ ID NO: 12), where the GnTIII catalytic domain is localized through the ManII domain responsible for the localization of the Golgi complex, unlike other nucleic acids encoding GnTIII activity used here , leads mainly to the appearance of branched non-fucosylated and branched hybrid structures (note the peaks with m / z 1664, 1810, 1826 and 1973 in Fig. 4). Therefore, in comparison with wild-type GnTIII and GnTI-GnTIII fused (SEQ ID NOS: 14 and 15), the fused ManII-GnTIII was more efficient in the synthesis of branched unfucosylated and branched hybrid oligosaccharides attached to the Fc region (compare peak ratios with m / z 1664, 1810, 1826 and 1973 to the general in Fig. 2, Fig. 3 and Fig. 4). The fractions of the branched non-fucosylated oligosaccharides of the Fc region obtained by expression of nucleic acids encoding wild-type GnTIII, GnTI-GnTIII fusion protein and ManII-GnTIII fusion protein were approximately 4, 13 and 33%, respectively. Branched oligosaccharides were not detected in the unchanged (non-glyco-modified) antibody.

An increase in the expression of the ManII-GnTIII fusion construct in antibody producing cells led to a further increase in the proportion of branched non-fucosylated oligosaccharides. This was demonstrated by the expression of the ManII-GnTIII construct from the vector (pETR1519) containing episi regulation oriP in transfected HEK-ENBA cells. This expression system is known to produce high levels of expression; it has also been used to express antibody genes from the pETR1520 vector. The spectrum of oligosaccharides of the purified unaltered (non-glyco-modified) antibody expressed in this system is shown in FIG. 5, which again shows a typical spectrum of oligosaccharides with peaks of unbranched fucosylated oligosaccharides containing 0.1 and 2 galactose residues (for example, compare Fig. 1 and Fig. 5., which show similar spectra of oligosaccharides of unchanged antibody expressed at higher levels in HEK-ENBA cells). Modification of antibody-producing cells with a nucleic acid encoding the ManII-GnTIII fusion protein expressed at higher levels in this system results in antibodies in which the majority of the Fc region-linked oligosaccharides were branched unfucosylated (see Fig. 6, in which the peaks branched non-fucosylated hybrid oligosaccharides with m / z 1664 and 1826 account for 90% of all oligosaccharides).

As mentioned above, endoglycosidase N (EndoH) was used to confirm the correspondence of branched non-fucosylated and branched hybrid structures to the different oligosaccharide peaks observed on MALDI spectra. The MALDI / TOF-MS spectra of neutral oligosaccharides formed by PNGaseF glycosidase and PNGaseF + EndoH glycosidases digested from glycans derived from anti-CD20 chimeric IgG1 class antibodies produced by HEK293 cells, glyco-modified by increased GnTIII expression, are shown in Fig. 2 (M2). 7. The peak at m / z 1664 can be mapped to two different glycans, namely a non-fucosylated branched hybrid and a non-fucosylated complex branched. Different structures exhibit the same m / z ratio due to the same composition of monosaccharides (Fig. 8B).

The cleavage of the released glycosidase PNGaseF of oligosaccharides with endolgicosidase H gives rise to new structures with the main peak shifted from m / z 1664 to m / z1460 (Fig. 7B). The difference corresponds to the mass of the N-acetylglucosamine residue. As mentioned above, endoglycosidase H is not capable of cleaving complex type oligosaccharides. Consequently, after cleavage by endoglycosidase H, the main peak with m / z 1664 can be mapped to the hybrid branched non-fucosylated oligosaccharides.

Other peaks can be mapped to complex or hybrid branched glycans. After cleavage by endoglycosidase H, the peak with m / z 1810 disappeared, which suggests that these structures are of the fucosylated branched hybrid type. Removal of one GlcNAc residue and one fucose residue (from fucosylated alpha 1-6 cor-fucosyltransferase of the GlcNAc residue at the reducing end) from the peak with m / z 1810 gives rise to a structure with m / z 1460. The disappearance of the peak with m / z 15060 after cleavage by endoglycosidase H (GlcNAc residue removed) and the appearance of a peak with m / z 1298 demonstrate the fact that peak 1502 can be mapped to oligosaccharides of the type of non-fucosylated branched hybrid. The disappearance of the peak with m / z 1647 after cleavage by endoglycosidase H demonstrates that this peak corresponds to a fucosylated hybrid branched structure. Removal of the GlcNAc residue and one fucose residue gives rise to a structure with m / z 1298. The peak with m / z 1826 corresponding to the type of unfucosylated hybrid branched disappeared after cleavage of Endo H. This gave rise to a structure with m / z 1622. After cleavage by endoglycosidase H, the peak is m / z 1053 can be assigned to oligosaccharides with a high mannose content (m / z 1257) cleaved by endoglycosidase N. Cleavage by endoglycosidase H, as expected, does not affect the peak on m / z 1689 (complex branched). As a result, based on the data in Table 1., we conclude that 88% of the oligosaccharide structures carry a branched "GlcNAc, of which 68% are non-fucosylated branched oligosaccharide structures, 22% are fucosylated hybrid branched and 6% are fucosylated complex branched oligosaccharide structures.

Mass balances (in molar fractions,%):

a) Peaks at m / z 1502 and 1647: 7 + 7% = 14% (expected), EndoH cleavage for both peaks gives rise to a peak at m / z 1298 (after processing EndoH, 13% was obtained)

b) Peaks at m / z 1664 and 1810: 47 + 13% = 62% (expected), EndoH cleavage produces a peak at m / z 1460 (after processing EndoH, 60% is obtained)

c) Peaks at m / z 1826 and 1972: 4 + 3% = 7%, Splitting EndoH generates a peak at m / z 1622 (7%)

Summary: Overall relative percentage of structures

bearing "branching" GlcNAc: 88% Unbranched hybrid branched 60% Branched Fucosylated Hybrid 22% Fucosylated Complex Branched 6%

The above data (Fig. 1 - Fig. 6) show that both the level of GnTIII expression and the specific localization domain, which is used to direct the GnTIII catalytic domain to the Golgi complex, affect the competition between

the catalytic domain of recombinant GnTIII, endogenous core-alpha-1,6-fucosyltransferase, ManII and GnTII enzymes for GnTI-modified oligosaccharide substrates. Higher expression provides GnTIII an advantage in this competition, leading to increased levels of branched hybrid and branched non-fucosylated oligosaccharides, as well as a concomitant decrease in the levels of branched complex and branched fucosylated oligosaccharides. This was previously noted also for wild-type GnTIII (Umana, P. et al., Nature Biotechnol. 17: 176-180 (1999)). Although, despite the fact that localization of the GnTIII catalytic domain through the GnTI or ManII localization domain leads to a similar (with wild-type GnTIII) overall level of branched oligosaccharides, it led to more effective competition with endogenous core-alpha compared to wild-type GnTIII -1,6-fucosyltransferase, as well as ManII and GnTII enzymes for modified GnTI oligosaccharide substrates.

The higher efficiency of the GnTI-GnTIII fusion protein in the synthesis of branched hybrid and branched non-fucosylated oligosaccharides in comparison with wild-type GnTIII can be explained by the earlier position (distribution) of GnTI relative to GnTIII in the Golgi complex in the direction of cis-trans transport of the substrate. The exact distribution in the Golgi complex of GnTI and ManII was previously determined by the method of quantitative immunoelectronic microscopy (Rabouille, C. et al. J Cell Sci. 108: 1617-27 (1995)). Both enzymes are distributed together along the Golgi complex, localized mainly in the medial (middle) and trans-tanks of the Golgi stack, and their level in the middle tanks is higher relative to the trans-tanks. The exact quantitative distribution of core-alpha-1,6-fucosyltransferase and wild-type GnTIII has not yet been determined. The above, however, does not explain why the fused ManII-GnTIII is significantly more efficient in the synthesis of branched hybrid and branched non-fucosylated oligosaccharides than the fused GnTI-GnTIII, since the spatial distribution of GnTI and ManII in the subcompartments of the Golgi complex is identical.

The higher efficiency of the ManII-GnTIII fusion protein indicates the existence of relatively organized functional reaction glycosylation subcompartments within the physical subcompartments of the Golgi complex medial and trans-cisterns. It is believed that the so-called “medial Golgi glycosylation enzymes”, GnTI, GnTII and ManII, exist in the Golgi complex in the form of high molecular weight complexes. However, if the localization domains allow these enzymes to form part of these complexes, the same should happen with

fused GnTI-GnTIII and ManII-GnTIII. The expression of recombinant GnTI-GnTIII fusion proteins did not lead to a change in the position of the wild-type GnTI enzyme, which was insignificant for the synthesis of Fc-region oligosaccharides, since all GnTIII constructs used here led to the majority of oligosaccharides being modified as GnTI-catalyzed reactions and reactions catalyzed by GnTIII.

Our data indicate that, owing to the properties of the ManII localization domain, finer structuring (combining) of the catalytic domains of endogenous GnTI and the recombinant fusion protein ManII-GnTIII occurs. It is known that organized structuring of enzymes that catalyze sequential reactions of a biosynthesis pathway, facilitating the transition of the products of the first reaction to the second catalytic site (relative to the diffusion removal of these products from enzymes), occurs in other biosynthesis pathways, such as glycolysis and polypeptide synthesis. There are reports that GnTI and ManII form “related oligomers” (kin oligomers), at least when one of these fragments moves along the endoplasmic reticulum (Nilsson, T. et al. EMBOJ 13 (3). 562-74 ( 1994)). It was found that a pair of charged amino acid residues in the core (framework) regions of these two enzymes play a key role in this “cognitive recognition”. Residues in GnTI carry a charge opposite to that of ManII residues. We have identified similar residues in the equivalent positions of the stem regions of other glycosylation enzymes of the Golgi complex involved in this part of the biosynthesis of N-linked oligosaccharides, namely core-alpha-1,6-fucosyl transferase (the same as for GnTI charges , instead of their complementary charges ManII), ManI and GnTII. We also found that these residues are conserved in different species. Although it has been suggested that these residues are not necessary for incorporation into high molecular weight complexes formed by enzymes, and even for localization in the Golgi complex (Opat, AS et al. J. Biol. Chem. 275 (16): 11836-45 (2000 )), they are possibly involved in a finer structuring of the catalytic domains during the biosynthesis of oligosaccharides. Such structuring is not necessarily irreversible; it can be mediated by temporary, dynamic interactions between enzymes. Perhaps there are additional determinants of structuring in other positions of the rod or catalytic region. However, any contribution of the ManII catalytic domain to the specific structuring of GnTI-ManII will be lost in the recombinant fusion polypeptide carrying the GnTIII catalytic domain.

FIG. 9 - FIG. 11 demonstrate increased antibody-dependent cellular cytotoxicity (ADCC), resulting from increased expression of a nucleic acid encoding a polypeptide with GnTIII activity, which is localized in the Golgi complex using various localization domains in antibody-producing cells. Increased ADCC resulting from increased expression of a recombinant polypeptide with GnTIII activity, which is localized in the Golgi complex using the domain responsible for localization in the Golgi complex of GnTI, is shown in FIG. 9. The spectrum of oligosaccharides of the control antibody used in the ADCC assay (FIG. 9) is shown in FIG. 1. The spectrum of glyco-modified antibodies oligosaccharides used in the ADCC assay (FIG. 9) is shown in FIG. 3. Increased ADCC resulting from increased expression of a recombinant polypeptide with GnTIII activity, which is localized in the Golgi complex using the ManII glycosidase domain responsible for the localization of the Golgi complex, shown in FIG. 10. The spectrum of oligosaccharides of the control antibody used in the ADCC assay (FIG. 10) is shown in FIG. 5. The spectrum of glyco-modified antibody oligosaccharides used in the ADCC assay (FIG. 10) is shown in FIG. 6.

In FIG. 11 shows that the expression of a recombinant polypeptide with GnTIII activity localized in the Golgi complex with the help of the localization of the ManII domain in the Golgi complex leads to an increase, compared with the wild-type GnTHI polypeptide, which has its own domain responsible for localization in the Golgi complex, ACCC activity. The spectrum of oligosaccharides, an antibody glycomodified by expression of wild-type GnTIII and used in the ADCC assay (FIG. 11), is shown in FIG. 2. The spectrum of oligosaccharides of an antibody glycodified by expression of a fusion polypeptide with GnTIII activity localized in the Golgi complex using the ManII localization domain and used in the ADCC assay (Fig. 11) is shown in Fig. 4. These data also show that antibodies with branched oligosaccharides, including branched hybrid and branched non-fucosylated oligosaccharides, have an increased activity of ADCC compared to antibodies with complex fucosylated unbranched oligosaccharides. It should be noted that all branched oligosaccharides of the more active antibody used in the ADCC assay, the results of which are shown in FIG. 10 are branched non-fucosylated hybrid oligosaccharides. As mentioned earlier, the use of a fusion polypeptide with GnTIII activity localized in the Golgi complex using the ManII localization domain leads to a more efficient synthesis of non-fucosylated branched oligosaccharides; in FIG. 11 shows that antibodies with elevated levels of these branched non-fucosylated oligosaccharides are more active than antibodies with lower levels of these oligosaccharides in ADCC reactions. An increase in the activity of ADCC is associated with an increase in the proportion of branched non-fucosylated oligosaccharides in the population of oligosaccharides associated with the Fc region, and a significant increase in activity is observed when this fraction increases by an amount greater than 15-20%.

Natural killer cells (NK cells) are known to be important participants in ADCC. On the surface of these cells is the activating receptor Fcgamma IIIA, also known as CD16a. The binding of the Fc region of antibodies associated with target cells with FcgammaRIIIA receptors on the surface of NK cells is necessary for the cross-linking of these receptors on NK cells and subsequent induction of ADCC. Thus, it is important to evaluate the binding of antibodies obtained by the methods described here to Fc receptors, especially to receptors in their native form, i.e. in the form in which they exist on the surface of the effector cells of the human immune system .. FIG. 12 shows that glycogen-modified antibodies obtained by expression in antibody-producing cells of a nucleic acid encoding a fusion polypeptide with GnTIII activity have increased binding affinity for the human FcgammaRIIIA activating Fc receptor. As mentioned above for cases of ADCC analysis, the level of branched non-fucosylated oligosaccharides in these antibodies is increased, which is the result of expression of the fusion polypeptide with GnTIII activity in antibody-producing cells. The NK cells used in this assay were obtained from a donor with a genotype that does not express the FcgammaRIIc receptor in donor NK cells (Metes, D. et al. J. Immunol. Methods 258 (1-2): 85-95 (2001) ) Thus, the only Fc receptor on the surface of such cells is the activating receptor FcgammaRIIIA. In FIG. 13 shows that a binding assay determines the affinity of specific binding to this receptor. This illustrates competition with a fragment of an antibody specifically blocking FcgammaRIIIA (Fab2 fragment of 3G8).

A convincing evidence of the strong influence of enhanced interactions between the Fc region and Fc receptors on the treatment of tumors with antibodies was the relationship between the patient’s homozygosity for the gene variant encoding the FcgammaRIIIA receptor with higher activity (the “high affinity” variant) found in patients receiving rituximab (Cartron , G. et al. Blood 99 (3): 754-8 (2002)). This was the only parameter for which a relationship was found with a significant increase in the objective degree of response and an increase in the proportion of molecular responses. The source of increased, due to enhanced interactions of FcgammaRIIIA-Fc, efficacy may be functions performed by various types of cells of the immune system, including natural killer cells (NK), macrophages and dendritic cells. Cross-linking the activating FcgammaRIIIA receptor on the surface of NK cells leads to tumor cell lysis by the ADCC mechanism, which many consider to be the main Fc-R-dependent in vivo cell lysis mechanism (Maloney, DG et al. Semite. Oncol. 29 (1 Suppl. 2 ): 2-9 (2002), Amigorena S., J Exp. Med. 195 (1): Fl-3 (2002)), as well as antibody-dependent cell phagocytosis (Hazenbos, WL et al. J. Immunol. 161 ( 6): 3026-32 (1998), Reff, ME, Heard C., Crit Rev Oncol Hematol. 40 (1): 25-35 (2001)) and the release of cytokines near tumor cells (Carson, W.E. et al Eur. J. Immunol. 31: 3016-3025 (2001)). These cytokines can, in turn, cause a direct cytolytic effect directed at the tumor cells, an anti-angiogenic effect, which leads to inhibition of tumor growth due to the cessation of its supply of oxygen and nutrients, as well as to increased presentation of tumor antigens, which is part of the active T-cell an immune response directed against tumor cells. Dendritic cells play a decisive role in the presentation of antigens to T cells, and cross-linking of FcgammaRIIIA on their surface (for example, antibodies bound by dying tumor cells previously attacked in vivo by the ADCC mechanism) can lead to an increase in the rate of dendritic cell maturation, absorption of antigens and their presentation T cells and cross-priming cytotoxic T cells, the latter potentially being a very effective mechanism for activating antitumor immunity (AmigorenaS., J. Exp.Med. 195 ( 1): F1-3 (2002), Kalergis, AM & Ravetch, JVJ Exp. Med. 195 (12): 1653-1659 (2002), Selenko, N. et. AU. Clin. Immunol. 22 (3): 124-130 (2002)). Cross-linking target cell antibodies with Fc receptors on the surface of the effector cells of the immune system can also lead to increased direct lysis of target cells, for example, by apoptosis induced by antibody-mediated cross-linking of target antigen molecules (Reff, M.E. . & Heard, C. Crit Rev Oncol Hematol. 40 (1): 25-35 (2001), Cragg, MS et al. Blood 101 (3): 1045-1052 (2003)). Of all the effector cells of the immune system, only NK cells possess exclusively activating surface Fcgamma receptors. In other types of cells, activating FcgammaRIII receptors are present simultaneously with the inhibiting receptor FcgammaRIIb, and the induction of antitumor effector functions is the result of the prevalence of activating signals over inhibitory ones.

In FIG. 15 shows that increased binding of the Fc receptor is selective for the activating receptor compared to the inhibitory FcgammaRIIb. Such selectivity is important, as explained above, for the cells of the immune system, such as NK cells, to perform their effector functions. Moreover, the increase in binding achieved by glyco-modification of the Fc region of the antibody by the methods described here is much greater than that usually observed in patients with high affinity FcgammaRIIIA homozygous variant / donor receiving a standard, unchanged antibody (Fig. 16), which the researchers associated with increased activity anti-cancer antibodies (Cartron, G. et al. Blood 99 (3): 754-8 (2002)).

The binding domain of the activating FcgammaRIIIB receptor is almost completely identical to the binding domain of FcgammaRIIIA. Therefore, the above data also indicate that the glyco-modified antibodies described here can cause an increase in effector functions mediated by effector cells that have FcgammaRIIIB on their surface, such as polymorphonuclear cells, including the release of toxic products and phagocytosis ((Reff, M.E. ., Heard, C. Crit Rev Oncol Henaatol. 40 (1): 25-35 (2001), Daeron, FM. Annu. Rev. Immunol. 15: 203-34 (1997), Ravetch, JV, Bolland S. Annu Rev. Immunol. 19: 275-90 (2001)).

In FIG. Figure 18 shows the spectrum of oligosaccharides of an anti-CD20 antibody produced by BHK cells growing in suspension and modified for high level constitutive expression of both a recombinant antibody and a fusion polypeptide with GnTIII activity. The oligosaccharide spectrum shows an increase in the levels of branched non-fucosylated and branched hybrid oligosaccharides in an antibody obtained in a cell modified with a GnTIII fusion protein (See also Table 2). These structures were not found in non-glyco-modified antibodies produced by non-glyco-modified BHK cells (See. Fig. 1). Modified cells expressing the GnTIII fusion construct showing normal growth in suspension and good antibody production productivity.

The relative percentage of different oligosaccharides of glyco-modified monoclonal antibodies produced by the cells of the stable cell line BHK-1502-28-11 are presented in Table 2.

Total branched: 88.6% (4 + 5 + 6 + 7 + 8 + 9 + 10 + 11 + 12)

Total non-fucosylated branched: 37.8% (4 + 5 + 7 + 9)

Total fucosylated branched: 50.8% (6 + 8 + 10 + 11 + 12)

Complex Branched: 17% (6 + 7 + 10 + 12)

Hybrid Branched: 71.6% (4 + 5 + 9 + 11

Analysis of oligosaccharides showed that 88.6% of the structures carry a branching residue of N-acetylglucosamine, 50.8% are fucosylated, and 37.8% are not fucosylated. Cleavage by endoglycosidase H of the released glycosidase PNG-ase demonstrated that most peaks were obtained from hybrid branched-type oligosaccharides (FIG. 19). FIG. Figure 20 shows the increased binding affinity of the glyco-modified antibody produced by cells of the BHK-1502-28-11 cell line with the activating FcgammaraIIIA Fc receptor on the surface of human NK cells. Cell lines growing in suspension and providing constitutively stable gene expression of both the antibody and the GnTIII fusion polypeptide are ideal for large-scale production of therapeutic antibodies. Using standard methods for cell modification, glycomodification can be performed either by introducing a GnTIII fusion protein gene into cell lines containing antibody genes, or by introducing antibody genes into cell lines containing GnTIII fusion protein genes (“pregly modified cell line”), or by simultaneously introducing antibody genes and a GnTIII fusion.

The anti-CD20 antibody produced in cells modified for high-level expression of a nucleic acid encoding a fusion polypeptide with GnTIII activity, localized in the Golgi complex using the ManII localization domain, was also investigated for complement-mediated lysis (Zd.-KOL) - another effector function that is not associated with Fc receptors on the effector cells of the immune system. The vast majority of oligosaccharides of this glyco-modified antibody were branched hybrid non-fucosylated oligosaccharides. These anti-CD20 antibodies showed reduced, compared with unchanged antibodies, complement-mediated lysis activity (Fig. 21). For some applications, it may be desirable to have antibodies with increased ADCC, but reduced complement-mediated lysis, for example, to reduce side effects such as those caused by complement-mediated lysis of blood vessel vasculitis at tumor sites. Another significant KOL-mediated side effect was observed during treatment with anti-CD20 antibody (van der Kolk L.E. et al. Br J Haematol. 115 (4): 807-11 (2001)). However, the above spectra of oligosaccharides also show that it is also possible to modify antibody-producing cells to express GnTIII fusion polypeptide at an average level, which leads to average levels of branched hybrid non-fucosylated oligosaccharides (above 15%), but with a significant proportion of complex oligosaccharides in the population associated with The Fc region of a glyco-modified oligosaccharide antibody. Such complex oligosaccharides are associated with normal, not lower, levels of KOL. Therefore, the data indicate that antibodies with increased ADCC can be produced in such a way that the COAL is maintained at the same level as that of unmodified antibodies.

The second chimeric IgG2 class antibody, C225, also known as cemximab, which recognizes the human epidermal growth factor (EGFR) receptor, has been glyco-modified by the methods described herein. In FIG. 22 shows the spectra of oligosaccharides of unmodified anti-EGFR antibody C225 and a glycomodified variant of the same antibody. The latter was produced by cells expressing a nucleic acid encoding a fusion polypeptide with GnTIII activity and localized in the Golgi complex using the mannosidase II localization domain. In FIG. 23 shows an anti-REFR antibody enhanced as a result of a modification of ADCC. Glyco-modified antibodies obtained by the methods described here and possessing increased ADCC and increased binding affinity of activating Fc receptors are promising molecules for the treatment of cancer and autoimmune diseases using antibodies, since they should lead to increased efficacy relative to the corresponding unchanged (non-glyco-modified) variants of the same antibodies, such therapy. Additionally, it should be possible to reduce therapeutic doses of glycogen-modified antibodies compared to unchanged, which will positively affect the economic aspects of antibody production.

EXAMPLE 2

Treatment of immune mediated thrombocytopenia in a patient with chronic graft rejection disease (“Graft-versus-host” disease).

Autoimmune thrombocytopenia in chronic graft rejection disease is an example of B-lymphocyte-related deregulation leading to a clinical disease. For the treatment of immune-mediated thrombocytopenia in a patient with chronic transplant rejection disease, a chimeric anti-CD20 monoclonal antibody prepared by the methods of the present invention and having increased ADCC is administered to the patient as described in nRatanatharathom, V. et al, Ann. Intern. Med. 133 (4): 275-79 (2000) (the full contents of the source are incorporated by reference). Specifically, the patient is prescribed weekly antibody infusions for 4 weeks (375 mg / m ² ). Antibody therapy results in a marked decrease in the number of B lymphocytes in peripheral blood and a decrease in platelet-associated antibody levels.

EXAMPLE 3

Treatment of severe immune-mediated true erythrocytic aplasia and hemolytic anemia.

Immuno-mediated acquired true erythrocyte aplasia is a rare disorder often associated with other autoimmune events. For the treatment of acquired immune-mediated true erythrocyte aplasia, the patient is prescribed, as described in Zecca, M. Et al., Blood 12: 3995-97 (1997) (the full content of the source is incorporated into the description by reference), a chimeric anti-CD20 monoclonal antibody prepared by the methods of the present invention and having increased ADCC. Specifically, a patient with immune-mediated true erythrocyte aplasia and hemolytic anemia receives two doses of the antibody, 375 mg / m ^2, weekly. After therapy with the use of antibodies, substitution treatment with intravenous hemeglobin is started. This treatment leads to a significant decrease in the level of B-lymphocytes in the peripheral blood and a significant increase in the number of reticulocytes, accompanied by an increase in the level of hemoglobin.

EXAMPLE 4

Materials and methods

1. Construction of expression vectors of the GalT fusion protein

Vector for constitutive expression of GalT

To construct a GalT expression vector, GalT coding for DNA is amplified from a DNA library (Clonetech) by overlapping PCR reactions. Immediately after the stop codon of this gene against the direction of transcription (upstream), the C-terminal portion of the c-myc epitope is added for subsequent convenient determination of GalT by the Western blot method (PEQKLISEEDL amino acid sequence). After confirming the correct sequence, the GalT gene is placed under the control of the MPSV promoter and the synthetic rabbit beta-globin polyadenylation signal is added. The final GalT expression vector also contains a separate expression cassette for selection (selection). This cassette contains the puromycin resistance gene, also controlled by the MPSV promoter, and a synthetic rabbit beta globin polyadenylation signal.

Replacement of amino acids encoding the GalT localization domain with aminoxylots encoding the human GnTI localization domain.

The construction of this hybrid galactosyl transferase gene is carried out, for example, by overlapping PCR reactions, resulting in a plasmid containing the GnTI-GalT fusion protein gene under the control of the MPSV promoter and puromycin resistance cassette for selection.

Replacement of amino acids encoding the GalT localization domain with aminoxylots encoding the localization domain of human mannosidase II.

The GalT expression vector is constructed. The resulting plasmid contains the ManII-GalT fusion gene under the control of the MPSV promoter

Combination of ManII-GalT fusion protein fusion gene with Epstein-Barr virus OriP replication origin

A DNA fragment containing oriP is subcolline, as described in Example 1, into the ManII-GalT hybrid expression vector described above.

Combination of ManII-GalT fusion protein fusion gene with a truncated CD4 cell surface marker gene

The expression vector is modified in order to additionally express the truncated CD4 cell surface marker gene. Briefly, the expression cassette of the recombinant fusion protein of the ManII-GalT fusion protein is converted from monocistronic to bicistronic by insertion, in the direction “in the direction of transcription” from the stop codon of the ManII-GalT fusion protein gene, the polio virus IRES element and the subsequent cDNA encoding truncated human CD4 protein (containing the human CD4 leader sequence necessary for secretion, and the transmembrane and extracellular domains located behind it).

3. Transfection of mammalian cells with GalT fusion protein and antibody expression vectors Transfection of BHK cells

Cells in the exponential growth phase (90-95% viability) were cultured, harvested, and then transfected as described in Example 1. To obtain glyco-modified antibodies, the cells were co-transfected with two plasmids: one for expressing the antibody and the other for expressing the GalT fusion polypeptide, in the ratio 3: 1, respectively

HEK293-ENBA Cell Transfection

HEK293-ENBA cells were transfected in an exponential growth phase as described in Example 1. To obtain glycogen-modified antibodies, the cells were co-transfected with two plasmids: one for expression of the antibody and the other for expression of the GalT fusion polypeptide, in a ratio of 4: 1, respectively. On day 5 after transfection, the supernatant was collected, first centrifuged for 5 minutes at 1200 rpm, and then for 10 minutes at 4000 rpm, and stored at 4 ° C.

Obtaining stable cell lines expressing recombinant anti-CD20 antibody and fused GalT

Clone BHK-1502-28, constitutively expressing the monoclonal anti-SB20 antibody genes and the neomycin resistance gene, are transfected with an expression vector by electroporation. The vector allows the constitutive expression of the ManII-GalT gene and the truncated form of human CD4, the expression of the latter being independent of IRES. The vector also contains an expression cassette of the puromycin resistance gene. Puromycin-resistant clones were first screened in order to obtain a set of clones that integrated vector DNA into their chromosomes. The clones were then examined for expression of truncated CD4 (tCD4), which serves as a marker of expression level of the bicistronic expression unit of the ManII-GalT + tCD4 gene. Recombinant antibody production was verified by enzyme-linked immunosorbent assay.

Transfection and subsequent studies of the level of expression of tCD4 are carried out as described in Example 1.

4. Obtaining and purification of unchanged and glyco-modified antibodies.

In the case of BHK cells transfected with an expression vector or cotransfected with an antibody expression vector and a GalT fusion protein expression vector, the cells are cultured after transfection and the culture supernatant is harvested after 96 hours of cultivation. Depending on the expected productivity, several (10-15) electroporations are carried out with the same vector.

In the case of HEK293-EBNA cells transfected with an expression vector or subjected to co-transfection with an antibody expression vector and a GalT fusion protein expression vector, the culture medium is replaced with fresh culture medium about 16 hours after transfection, and fresh medium is collected after another 120 hours of culturing the transfected cells.

In the case of a stable BHK-1502-28 cell line, cultures were seeded at a concentration of 500,000 cells / ml and after 4 days of culture, the supernatant was collected.

Antibody purification

The monoclonal antibody is purified from the culture supernatants by two successive chromatography steps: protein A chromatography and cation exchange chromatography as described in Example 1.

5. Analysis of the oligosaccharide

Oligosaccharides were released from antibodies enzymatically: by PNGase glycosidase digestion, while the antibodies were either immobilized on the PVDF membrane or were in solution.

The resulting cleavage solution containing the released oligosaccharides was either immediately prepared for analysis on a MALDI / TOF-MS mass spectrometer, or subjected to further digestion with EndoH endoglycosidase before analysis for analysis on a MALDI / TOF-MS mass spectrometer

A method of releasing oligosaccharides for immobilized on a membrane PVDF antibodies and a Method of releasing oligosaccharides for antibodies in solution in solution is carried out as described in Example 1.

Application of EndoH glycosidase cleavage of PNGaseF-released oligosaccharides by glycosidase to establish correspondence between hybrid glycosylated structures of oligosaccharides and peaks of neutral oligosaccharides in the MALDI / TOF-MS spectrum

The oligosaccharides released by PNGaseF glycosidase are then digested with endoglycosidase H (EC 3.2.1.96) as described in Example 1.

MALDI / TOF-MS

Samples were prepared containing the enzymatic cleavage products of the released oligosaccharides, and then analyzed on a MALDI / TOF-MS mass spectrometer, as described in Example 1.

6. Preparation and isolation of cells

Peripheral blood mononuclear cells (PBMCs) are prepared using Histopaque-1077 (Sigma Diagnosticslnc, St. Louis, M063178 USA), strictly following the manufacturer's instructions and the protocol described in Example 1.

Human NK cells are isolated from PBMCs using the negative selection procedure as described in Example 1.

8. Analysis of the ACCC

MNPCs or NK cells, as effector cells, are prepared as described above and their ability to mediate cytotoxicity in antibody-dependent cellular cytotoxicity (ADCC) reactions is analyzed as described in Example 1.

9. FcgammaRIIIA binding to NK cells and FcgammaRIIb binding to Raji lymphoma cells.

The binding of FcgammaRIIIA on freshly isolated NK cells and the binding of FcgammaRIIb on Raji lymphoma cells are determined as described in Example 1.

10. Analysis of complement dependent cell cytotoxicity

A complement dependent cytotoxicity analysis is performed using antibody dilutions according to the methods described in Example 1.

Results and discussion

Assays were performed to demonstrate the effect of the expression of genes encoding GalT activity polypeptides on cell viability, cell growth, and antibody production relative to cells not producing such polypeptides. The glycosylation variants of purified antibodies were analyzed on a MALDI / TOF-MS mass spectrometer as described in Example 1. Using this technique, it is possible to determine the fractions of different types of oligosaccharides in the total, initial, oligosaccharide population associated with the Fc region, and it is also possible to determine the correspondence of structures different peaks of the spectrum (Umana, P. et al., Nature Biotechnol. 17: 176-180 (1999)).

The oligosaccharide spectrum of the unchanged antibody is determined. Specifically, it is determined whether the modification of antibody-producing cells by expression of wild-type GalT results in the appearance of mainly galactosylated complex two-antenna oligosaccharides with fucosylated cortex. It is also determined whether the modification of antibody producing cells by expression of a nucleic acid encoding a GnTI-GalT fusion polypeptide, where the GalT catalytic domain is localized by the GnTI domain, responsible for localization in the Golgi complex, leads to the appearance of mainly galactosylated complex two-antenna oligosaccharides, compared with wild-type GalT. If galactosylated non-fucosylated and galactosylated hybrid structures are synthesized, this may be the result of competition between GalT and other glycosyltransferases or glycosidases. It is expected that an oligosaccharide modified during the GalT-catalyzed GalT-catalyzed reaction by the addition of a galactose residue cannot be further modified by alpha-1,6-cor-fucosyltransferase, alpha-mannosidase II (ManII) of the Golgi complex and GnTII of the Golgi complex.

In order to estimate the proportion of galactosylated non-fucosylated and galactosylated hybrid oligosaccharides attached in the Fc region, cleavage with endoglycosidase H, which “distinguishes” hybrid and complex oligosaccharides, is used.

Tests are carried out to determine whether the modification of antibody-producing cells by expression of a nucleic acid encoding the ManII-GalT fusion polypeptide, where the GalT catalytic domain is localized in the Golgi complex using the ManII domain, results in the appearance of mainly galactosylated non-fucosylated and galactosylated hybrid oligosaccharides . In particular, it is determined whether the fused ManII-GalT is more efficient than the wild-type GalT and the GnTI-GalT hybrid in the synthesis of the attached Fc regions of galactosylated non-fucosylated and galactosylated hybrid oligosaccharides.

As mentioned above, endoglycosidase H (EndoH) is used to confirm the correspondence of galactosylated non-fucosylated and galactosylated hybrid structures to the different peaks of oligosaccharides observed in the MALDI spectra. The percentage of non-fucosylated hybrid structures, fucosylated hybrid structures and fucosylated complex oligosaccharide structures, fucosylated hybrid and fucosylated complex oligosaccharide structures among the oligosaccharide galactose residues that carry the galactose residue is determined.

The influence of the GalT expression level, and in particular the localization domain used to direct the GalT catalytic domain to the Golgi complex, on the competition between the recombinant GalT catalytic domain and the endogenous core-alpha-1,6-fucosyl transferase, ManII and GnTII for GalT-modified oligosaccharide substrates is determined. The level of antibody-dependent cellular cytotoxicity (ADCC), which is the result of increased expression in an antibody-producing cell of a nucleic acid encoding a GalT activity polypeptide localized in the Golgi complex by different localization domains, is determined.

It is also determined whether glyco-modified antibodies produced by expression in antibody producing cells have a nucleic acid encoding a fusion polypeptide with GalT activity, increased binding activity to the human FcgammaRIIIA activating Fc receptor or to the FcgammaRIIb inhibitory receptor.

GalT constructs can suppress the activity of endogenous core cortex-1,6-fucosyltransferase by glyco-modifying the Fc region of the antibody and, consequently, increasing ADCC.

The spectrum of oligosaccharides of the anti-CD20 antibody obtained in BHK cells growing in suspension and modified for high-costitative expression of both a recombinant antibody and a fusion polypeptide with GalT activity is determined. Also determine the relative percentage of oligosaccharides of glycol-modified monoclonal antibodies produced by cells of the stable cell line BHK-1502-28-11.

EXAMPLE 5 Materials and Methods

1. Construction of expression vectors ManII and GnTII

To construct the expression vector of mannosidase II (SEQ ID NO: 17), cDNA was subcloned into the plasmid (expression vector) in the direction “in the direction of transcription” from the MPSV promoter and in the direction “opposite to the direction of transcription” from the synthetic rabbit beta-globin polyadenylation signal. For expression of GnTIII, a plasmid (expression vector) was used with human GnTII cDNA subcloned in the direction “in the direction of transcription” from the promoter / enhancer of human cytomegalovirus and in the direction “opposite to the course of transcription” from the bovine growth hormone polyadenylation signal.

The combination of expression vectors with the point of origin of replication of OriP Epstein-Barr virus.

The OriP DNA fragment was subcloned, as described in Example 1, into the ManII expression vector described above. The result was the expression vector ManII pCLF9. The DNA fragment with OriP was Subcloned, as described in Example 1, into the GnTII expression vector described above. As a result, a GnTII pGnTII expression vector was obtained.

2. Transfection of HEK293-ENBA cells

HEK293-ENBA cells were transfected in an exponential growth phase as described in Example 1. To obtain an unchanged Cwt antibody, cells were transfected with an antibody expression vector (pETR1520). To obtain the glycogen-modified Cbrt antibody, the cells were co-transfected with two plasmids: one for expression of the antibody (pETR1520) and the other for expression of the GnTIII fusion polypeptide (pETR1519), in a 4: 1 ratio, respectively. To obtain the glycomodified Cm antibody, the cells were co-transfected with three plasmids: one for expression of the antibody (pETR1520), one for expression of the GnTIII fusion polypeptide (pETR1519), and another for the expression of mannosidase II (pCLF9) in a ratio of 3: 1: 1, respectively . To obtain the glycomodified Cmg antibody, the cells were co-transfected with four plasmids: one for expressing aHTHTena (pETR1520), one for expressing the GnTIII fusion polypeptide (pETR1519), one for expressing mannosidase II (pCLF9) and another one for expressing GnTII (pnFII) ( 4: 0.33: 0.33: 0.33 ratio, respectively.

3. Obtaining and purification of unchanged and glyco-modified antibodies

The culture medium of the transfected cells described above was replaced with fresh culture medium at approximately the 16th hour after transfection of the cultivation, fresh medium was collected after another 120 hours of cultivation. The collected supernatant was centrifuged for 5 min at 1200 rpm, after which it was centrifuged a second time (4 min at 4000 rpm). The supernatant was stored at 4 ° C.

Antibody purification

Monoclonal antibodies were purified from culture supernatants using two sequential chromatography steps: Protein A chromatography and cation exchange chromatography as described in Example 1. For cwt7, cbrt5 and cm1 antibodies, extrusion chromatography on a Superdex 200 column (Amersham Pharmacia) was additionally performed, phosphate added buffer solution after the stage of cation exchange chromatography and the peak of monomeric antibodies was collected.

4. Analysis of oligosaccharides

Oligosaccharides were released from antibodies in solution enzymatically: by PNGaseF glycosidase digestion, as described in Example 1. The use of EndoH glycosidase digestion of PNGaseF glycosidase-released oligosaccharides with oligosaccharide oligosaccharides M DF Ms

The oligosaccharides released by PNGaseF glycosidase are then digested with endoglycosidase H (EC 3.2.1.96) as described in Example 1.

MALDI / TOF-MS

Samples were prepared containing the enzymatic cleavage products of the released oligosaccharides, and then analyzed on a MALDI / TOF-MS mass spectrometer, as described in Example 1.

5. Preparation and isolation of cells

Peripheral blood mononuclear cells (PBMCs) are prepared using Histopaque-1077 (Sigma Diagnosticslnc, St. Louis, M063178 USA), strictly following the manufacturer's instructions and the protocol described in Example 1.

Human NK cells are isolated from PBMCs using the negative selection procedure as described in Example 1.

35

7. Analysis of the ACCC

MNPCs, as effector cells, are prepared as described above and their ability to mediate cytotoxicity is analyzed in the Antibody-Dependent Cell Cytotoxicity Assay (ADCC) as described in Example 1.

8. Binding of FcgammaRIIIA on NK cells

The binding of FcgammaRIIIA on freshly isolated NK cells and the binding of FcgammaRIIb on Raji lymphoma cells are determined as described in Example 1.

9. Analysis of complement dependent cytotoxicity

Analyzes of complement dependent cytotoxicity were performed using antibody dilutions according to the methods described in Example 1, followed by modification of the human complement source. Briefly, normal human serum (NPF) was prepared from the blood of healthy volunteers. Blood was allowed to coagulate for 1 hour, then centrifuged at 1200 g for 20 minutes. The cell-free supernatant was aliquoted and stored at -80 ° C. NPFs were used in relation to 20% of the final analysis volume.

Results and discussion

Glyco-modified variants of anti-CD20 chimeric IgG1 class antibodies (C2B8 chimeric antibody also known as rituximab) produced mammalian cell cultures co-transfected with antibody gene expression vectors and gene expression vectors encoding polypeptides with GnTIII and mannosidase II activity. An additional glyco-modified variant of the antibody was obtained in a culture of mammalian cells subjected to cotransfection with antibody gene expression vectors and gene expression vectors encoding polypeptides with GnTIII, mannosidase II and GnTII activity. To obtain the glycogen-modified Cbrt antibody, the cells were co-transfected with two plasmids: one for expressing the antibody (pETR1520) and the other for expressing the GnTIII polypeptide (pETR1519). To obtain the glycomodified Cm antibody, cells were co-transfected with three plasmids: one for expression of the antibody (pETR1520), one for expression of the GnTIII polypeptide (pETR1519), and another for expression of mannosidase II (pCLF9). To obtain the glycomodified Cmg antibody, cells were co-transfected with four plasmids: one for expression of the antibody (pETRT520), one for expression of the GnTIII polypeptide (pETR1519), one for expression of mannosidase II (pCLF9) and one for expression of GnTII (pGnTII). An unmodified (non-glyco-modified) variant of the same antibody (“Cwt”) was obtained by transfecting mammalian cells solely with the antibody gene expression vector. Transfected cells were cultured for 5 days, then secreted recombinant antibodies were purified from the culture medium. Expression of genes encoding polypeptides with GnTIII and ManII activity did not significantly affect cell viability, cell growth or antibody production compared to cells not producing such polypeptides with glycosyltransferase or glycosidase activity.

The object of analysis was the glycosylation pattern of purified antibodies. These antibodies carry N-linked oligosaccharides attached exclusively to the Asn297 residue of the human IgG1 Fc region. Oligosaccharides were separated from antibodies by enzymatic means (using PNGase glycosidase digestion) and then examined on a MALDI / TOF-MS mass spectrometer. Using this technique, it is possible to determine the various fractions of oligosaccharides in the total, initial, population of oligosaccharides associated with the Fc region, as well as to establish the correspondence of structures to different peaks of the spectrum (Umana, P. et al., Nature Biotechnol. 17: 176-180 (1999 )).

In FIG. 26 shows a spectrum of neutral oligosaccharides of an unchanged recombinant anti-CD20 chimeric IgG1 C2B8 Cwt antibody obtained on a MALDI / TOF-MS mass spectrometer. As for the unaltered antibody described previously in Example 1, FIG. 5, Cwt had a typical spectrum of oligosaccharides with peaks at m / z 1485, 1648, and 1810 and consisted of bi-antenna corfucosylated complex oligosaccharides with the number of galactose residues equal to 0, 1, and 2, respectively. Modification of antibody producing cells by expression of a nucleic acid encoding a ManII-GnTIII fusion polypeptide in which the catalytic domain is localized in the Golgi complex using the ManII localization domain resulted in antibody production (Cbrt), in which the majority of the Fc region-linked oligosaccharides were branched unfucosylated hybrid (see Fig 27). As described in Example 1, endoglycosidase H (EndoH) was used to confirm the correspondence of galactosylated non-fucosylated and galactosylated hybrid structures to the different peaks of oligosaccharides observed in the MALDI spectra.

Modification of antibody-producing cells by co-expression of a nucleic acid encoding a ManII-GnTIII fusion polypeptide in which the catalytic domain is localized in the Golgi complex using the ManII localization domain, and the nucleic acid encoding ManII leads to the production of antibody (Cm), in which most Fc-linked oligosaccharides were branched unfucosylated complex (see FIG. 28). Modification of antibody producing cells by co-expression of a nucleic acid encoding a ManII-GnTIII fusion polypeptide in which the catalytic domain is localized in the Golgi complex using the ManII localization domain, as well as the nucleic acid encoding ManII and the nucleic acid encoding GnTII, resulting in antibody production (Cmg), in which the majority of the Fc-region-linked oligosaccharides are branched non-fucosylated complex, with the proportion of branched non-fucosylated complex oligosaccharides Fc-o Asti was even higher than the antibody cm.

Fig. 29 shows data demonstrating enhanced antibody-dependent cellular cytotoxicity (ADCC) resulting from the expression in antibody-producing nucleic acid cells encoding the ManII-GnTIII fusion polypeptide in which the catalytic domain is localized in the Golgi complex using the ManII localization domain, where the encoding ManII- The GnTIII nucleic acid is either expressed alone (Cbrt antibody) or coexpressed simultaneously with the nucleic acid encoding ManII (Cm antibody). Thus, an increase in the level of branched non-fucosylated hybrid or complex type oligosaccharides in the Fc region of glyco-modified antibodies leads to an increased activity of ADCC.

Natural killer cells (NK cells) are known to be important participants in ADCC. On the surface of these cells is the activating receptor Fcgamma IIIA also known as CD16a. The binding of the Fc region of antibodies associated with target cells with FcgammaRIIIA receptors on the surface of NK cells is necessary for the cross-linking of these receptors on NK cells and subsequent induction of ADCC. Therefore, it is important to evaluate the binding of antibodies obtained by the methods described here to Fc receptors, especially to receptors in their native form, i.e. in the form in which they exist on the surface of the effector cells of the human immune system .. FIG. 30 demonstrates that glyco-modified antibodies obtained by expressing a nucleic acid encoding a fusion polypeptide with GnTIII activity in antibody producing cells, where the GnTIII encoding nucleic acid is expressed either alone (Cbrt antibody) or coexpressed in the antibody producing cell simultaneously with the nucleic acid, encoding ManII (Cm antibody), have increased binding affinity for the FcgammaRIIIA human activating Fc receptor. As mentioned above for cases of ADCC analysis, the level of branched non-fucosylated oligosaccharides in these antibodies is increased, which is the result of expression of the fusion polypeptide with GnTIII activity in antibody-producing cells.

Consequently, an increase in the level of branched non-fucosylated hybrid or complex type oligosaccharides in the Fc region of m glyco-modified antibodies leads to an increased activity of ADCC. The NK cells used in this assay were obtained from a donor with a genotype that does not allow expression of the FcgammaRIIc receptor in the NK cells of the donor (Metes, D. et al. J. Immunol. Methods 255 (1-2): 85-95 (2001 )). Thus, the only Fc receptor on the surface of such cells is the activating receptor FcgammaRIIIA

The binding domain of the activating FcgammaRIIIB receptor is almost completely identical to the binding domain of FcgammaRIIIA. Therefore, the above data also indicate that the glyco-modified antibodies described here can cause an increase in effector functions mediated by effector cells that have FcgarnmaRIIIB on their surface, such as polymorphonuclear cells, including the release of toxic products and phagocytosis ((Reff, M. E ., Heard, C. Crit Rev Oncol Henaatol. 40 (1): 25-35 (2001), Daeron, FM. Annu. Rev. Immunol. 15: 203-34 (1997), Ravetch, JV, Bolland S. Annu Rev. Immunol. 19: 275-90 (2001)).

Cbrt, an anti-CD20 antibody produced by cells modified to express a nucleic acid encoding a fusion polypeptide with GnTIII activity, which is localized in the Golgi complex using the ManII localization domain, was also examined for complement-mediated lysis (Zd.-KOL), another effector function which is not associated with Fc receptors on the effector cells of the immune system. The vast majority of oligosaccharides of this glyco-modified antibody belong to the type of branched hybrid non-fucosylated oligosaccharides. In Cbrt antibodies, a reduced, compared with unchanged Cwt antibodies, complement-mediated lysis activity was observed (Fig. 31). For some applications, it may be desirable to have antibodies with increased ADCC, but reduced complement-mediated lysis, for example, reducing side effects, such as vasculitis in blood vessels at tumor sites, mediated by QOL. Another KOL-mediated side effect was observed during treatment with anti-CD20 antibody (van der Kolk L.E. et al. Br J Haematol. 115 (4): 807-11 (2001)). However, it is possible to obtain glycogen-modified antibodies with increased ADCC activity and FcgammaRTII binding without a significant decrease in the activity of KOL compared to unchanged antibodies, as is the case with Cm antibodies (Fig. 31). Such antibodies are desirable for applications where the maximum elimination of target cells requires both high ADCC and high complement activation and KOL activity. The above spectra of oligosaccharides show that it is possible to modify cells to produce antibodies in which the majority of oligosaccharides attached to the Fc region belong to the type of branched unfucosylated complex instead of branched unfucosylated hybrid. This can be achieved by coexpressing the GnTIII fusion polypeptide together with a nucleic acid encoding a ManII antibody Cm) or together with a nucleic acid encoding a Man II and a nucleic acid encoding a GnTII (Cmg antibody).

Glyco-modified antibodies have increased ADCC activity and increased FcgammaRTII binding activity, which is associated with increased levels of branched non-fucosylated oligosaccharides, while their KOL activity increased with an increase in the proportion of complex oligosaccharides relative to the fraction of hybrid oligosaccharides.

This and previous examples describe the expression of nucleic acid fusion coding polypeptides encoding, where the fusion polypeptides are localized in the Golgi apparatus and have a catalytic domain that competes with endogenous fucosyl transferases for oligosaccharide acceptors previously modified in the course of reactions catalyzed by GnTI. Recombinant glycoproteins produced by cells modified in this way have elevated levels of unfucosylated oligosaccharides. This example demonstrates that co-expression in such host cells of nucleic acids encoding ManII and / or GnTII simultaneously with the nucleic acids described above encoding fusion polypeptides leads to an increase in the biosynthesis of complex oligosaccharides due to hybrid oligosaccharides and, consequently, to an increase in the synthesis of glycoproteins with an increased, relative to the glycoproteins produced in cells that do not co-express nucleic acids encoding ManII and / or GnTII, the level of unfucosylated complex oligosaccharides.

EXAMPLE 6

Increased expression of alpha-mannosidase

Molecular cloning

Human Alpha Mannosidase II

The gene encoding human alpha-mannosidase II ("hManII") (EC 3.2.1.114, SEQ ID NO: 17) under the control of the MPSV promoter, was cloned into an expression vector containing the oriP element. The resulting pCLF9 expression vector is shown in FIG. 32A. The expression vectors encoding the light and heavy chains of the anti-CD20 monoclonal antibody were, respectively, the vectors pETR1842 and pETR1843 (Fig. 32B and Fig. 32C).

ManII-GalT Fusion Protein

A fusion protein (SEQ ID NO: 20) consisting of a CTS region (cytoplasmic + transpordal + "core" region) of 11 hours mannosidase and a catalytic domain of beta-1,4-galactosyltransferase (M22921, amino acids 126-397) was constructed as described below. The CTS region of the PC mannosidase was amplified by PCR from the vector pETR1484 (primers CF33, GAB252). The GalT catalytic domain (amino acids 126-397) using primers CF31 and CF35 from the pBlueGalT vector. The CTS region of IInh mannosidase was combined with the GalT catalytic domain, resulting in a fusion protein controlled by the MPSV promoter (pCLF24). The complete GalT gene was obtained from pBlueGalT. The GalT coding gene was sequenced (8E0 ID NO: 16):

MRLREPLLSGSAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSR

LPQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDS

SPWDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDL

ELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHP

VLQRQQLDYGIYVrNQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDV

DLIPMNDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLT

INGFPNNWGWGGEDDDIFNRLWRGMSISRPNAWGRCRMIRHSRDKK

NEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS

The 5 ′ portion of GalT was amplified with CF31 / CF38 primers and the FseI restriction site in front of the gene was added. Sequencing showed the correctness of the sequence; after which the sequence was translated into the vector pCLF24 by restriction enzyme digestion with FseI / DraIII, which gave the vector pCLF26. After pCLF24 and pCLF26 OriP were added to the vectors, the pCLF25 and pCLF27 vectors, respectively, were obtained (Fig. 33A and Fig. 33B).

Expression of alpha-mannosidase and ManII-GalT in HEK293-ENBA cells.

HEK293-ENBA cells were transfected with a calcium phosphate method. Briefly, for T150 transfection 24 hours before transfection, 15 million cells were plated in 28 ml of DMEM containing 10% fetal serum and 250 μg / ml neomycin and incubated at 37 ° C in an atmosphere with 5% CO ₂ overnight.

For each T150 vial to be transfected, a solution of DNA, CaCl _2, and water was prepared: 94 μg of total DNA of the plasmid vector were mixed, 469 μl of a 1M CaCl ₂ solution, and the mixture volume was adjusted to 938 μl by adding water. To this solution, 938 μl of a solution containing 50 mM HEPES, 280 mM NaCl and 1.5 mM Na ₂ HPO _4, with a pH of 7.05 was added and immediately stirred for 10 seconds, after which it was allowed to stand at room temperature for 20 seconds. The suspension was diluted with 24 ml of DMEM medium supplemented with 2% fetal serum and added to T150 instead of the existing medium. Cells were incubated at 37 ° C, 5% CO ₂ for approximately 17-20 hours, then the medium was replaced with 30 ml of DMEM containing 10% fetal serum.

To study the effect of alpha-mannosidase II on the competition of corfucosyl transferase, cells were transfected with the vectors pETR1842, pETR1843 and pCLF9 in a ratio of 2: 1: 1, respectively. To express the ManII-GalT fusion protein, cells were transfected with the vectors pETR1842, pETR1843 and pCLF25 in a ratio of 2: 1: 1, respectively. On the 5th day after transfection, the supernatant was collected, centrifuged for 5 minutes at 1200 rpm, and then centrifuged for a second time (4 minutes at 4000 rpm). The supernatant was filtered and stored at 4 ° C.

Purification of Monoclonal Anti-CD20 Antibody

The monoclonal antibody is purified from 30 ml of the supernatant by two chromatography steps: Protein A chromatography to separate the monoclonal antibody from the bovine antibody present in the serum, and cation exchange chromatography following it to replace the sample buffer with PBS.

Oligosaccharide Analysis

Glycosidase Cleavage PNGaseF

A monoclonal antibody sample (50 μg) was incubated with N-glycosidase F (recombinant, Roche, Switzerland) at a concentration of O.lmU / μl. Cleavage was carried out in 0.2 mM Tris buffer (pH 7.0) for 3 hours at 37 ° C. Then, the released oligosaccharides were incubated with 150 mM acetic acid for 3 hours at room temperature. Then, the salt concentration in the samples was reduced using 0.6 ml of a cation exchange resin (resin AG50W-X8, hydrogen form, granule size 100-200, BioRad, Hercules, California) enclosed in a Micro Bio-Spin chromatography column (BioRad, Hercules, California).

Cleavage by endoglycosidase N.

Before treatment with acetic acid, the released PNGaseF glycosidase was digested with endoglycosidase H (EC 3.2.1.96 Roche, Switzerland), an enzyme that cleaves N-linked oligosaccharides between the N-acetylglucosamine residues. This enzyme will split the "oligomannose" type glycans and most of the hybrid type glycans, while complex type oligosaccharides do not hydrolyze.

Oligosaccharides cleaved 0.2 mU / μl of endoglycosidase H in 2 mM Tris buffer. Cleavage was carried out in 0.2 mM Tris buffer (pH 7.0) for 3 hours at 37 ° C. Then, the released oligosaccharides were incubated with 150 mM acetic acid for 3 hours at room temperature. Then, the salt concentration in the samples was reduced using 0.6 ml of cation exchange resin (resin AG50W-X8, hydrogen form, granule size 100-200, BioRad, Hercules, California) enclosed in a Micro Bio-Spin chromatographic column (BioRad, Switzerland)

Preparation of matrix and samples.

A matrix based on 2,5-dihydroxybenzoic acid (sDHB) was prepared as follows: 2 mg of 2,5-dihydroxybenzoic acid and 0.1 mg of 5-methoxysalicylic acid were dissolved in 1 ml of a mixture of ethanol and 10 mm aqueous sodium chlorid (in a volume ratio of 1: 1 ) 1 μl of the sample was applied to a stainless steel target and mixed with 1 μl of a 2,5-dihydroxybenzoic acid matrix. Samples were air dried and 0.2 μl of ethanol was applied.

MALDI / TOF Analysis

To obtain mass spectra, we used a Voyager Elite MALDI / TOF mass spectrometer (Perspective Biosystems) with an extraction delay. Positive ions were accelerated at 20 kV after a delay of 57 ns. The unit worked in reflex mode. To obtain the final spectrum, five spectra of 40 strokes (200 strokes) were combined. To assign masses to ions, an external calibration with standard oligosaccharides was used.

The spectrum of oligosaccharides

The spectrum of oligosaccharides of the anti-CD20 antibody produced in the presence of ManII is shown in FIG. 34. Oligosaccharides associated with the Fc region of antibodies are complex structures, 48% of which lack bark fucose. Alpha-mannosidase II competes with corticosyltransferase, giving rise to 48% of non-fucosylated oligosaccharide structures. In the absence of alpha-mannosidase II, the oligosaccharides of the Fc region of the antibody are represented exclusively by fucosylated structures (wild-type antibody).

The spectrum of oligosaccharides of the anti-CD20 antibody produced in the presence of the ManII-GalT fusion polypeptide is shown in FIG. 35A-B. In the case of the ManII-GalT fusion protein, as well as for alpha-mennosidase II, the number of unfucosylated oligosaccharide structures increases. Among unfuxilated structures, there is a high percentage of structures with a high mannose content (m / z 1256, 1419 and 1581). In addition to these 67 percent of non-fucosylated Sugars, 30% of hybrid non-fucosylated structures were found (m / z 1622). Therefore, a sample produced in the presence of the ManII-GalT fusion protein exhibits an almost 100% content of non-fucosylated structures.

The biological activity of antibodies produced in the presence of alpha-mannosidase II or the fusion protein ManII-GalT

In order to determine the effect of the action of alpha-mannosidase II and the enzyme ManII-GalT in competition with corfucosyl transferase, corresponding biological analyzes were performed. Samples were tested for in vitro antibody-dependent cell cytotoxicity (ADCC) and CD 16 receptor binding expressed on the surface of the modified CHO cell line (CHO-1708-37).

IeG binding at CHO-1708-37

CHO-1708-37 cells expressed the FcγRTIIA receptor (CD16) and the FcγRI receptor γ chain on their surface. FcγRIIIA receptor expression (CD16) was evaluated using FACS analysis using a monoclonal antibody 3G8-FITC (Fig. 36). CHO-1708-37 cells were incubated at a concentration of 1 million / ml in an FBI containing 0.1% BSA, with different variants of antibodies at different concentrations (10, 3, 1, 0.3, 0.1 μg / ml) in triplicates. Cells were incubated for 30 minutes at 4 ° C and then washed with PBS containing 0.1% BSA. Antibody binding was determined by incubating it at a ratio of 1: 200 with a fluorescin isothiocyanin conjugate and a goat IgG fragment against a human F (ab ') fragment ₂ (JacksonlmmunoResearch, West Grove, PA) for 30 minutes at 4 ° C. The fluorescence intensity associated with the associated antibody variants was determined on a FASCalibur instrument (BD Bioscience, San Jose, PA) that transmits living cells.

The following antibody variants were included in the binding assay experiment:

Cwt 8 (wild type 1)

ManII (alpha-mannosidase II)

an antibody (ManII) produced in the presence of alpha-mannosidase II binds to the FcγRIIIA receptor with higher affinity than wild-type antibodies.

In Vitro Antibody Cellular Cytotoxicity (ADCC)

Peripheral blood mononuclear cells (PBMCs) were prepared using Histopaque-1077 (Sigma Diagnostics Inc., St. Louis, M063178, USA), strictly following the manufacturer's instructions. Briefly, from volunteers who ran for 1 minute at full speed to increase the percentage of natural killers (NK cells), venous blood was taken with heparin syringes. Blood was diluted in an FBI that did not contain Ca and Mg in a ratio of 1: 0.75-1.3, and layered on Histopaque-1077. The gradient was centrifuged at 400 × g without interruption for 30 minutes at room temperature (CT). Histopaque-1077 border adjacent fluid containing PBMC was collected, washed with PBS (50 ml per cell from two gradients) and collected by centrifugation at 300 xg for 10 minutes at RT. After resuspending the pellet at the FBI, the PBMCs were counted and washed a second time by centrifugation at 200 × g for 10 minutes at RT. Then the cells were resuspended in a suitable medium for subsequent procedures.

Target cells (Raji cells) were washed in the PBS, counted and resuspended at a concentration of 1 million cells per ml in DMEM containing 10% fetal serum and 1% Glutamax. To this was added calcium in a ratio of 1: 100, and then the cells were incubated for 30 min at 37 ° C. Then the cells were washed in the FBI, counted and resuspended at a concentration of 0.3 million cells per ml in AIM-V medium. Wells of a round-bottom 96-well plate were added in 100 μl of these cells. Antibodies were diluted in AIM-V medium, 50 μl was added to the target cells and left for 10 min at room temperature for binding. PBMC effector cells were prepared as described above. The ratio of effector cells to target cells was 25: 1. Effector cells were prepared at a concentration of 15 million cells per ml in AGM-V medium and introduced 50 μl into the wells. The plates were incubated for 4 hours at 37 ° C in a humid atmosphere containing 5% CO ₂ . Cells were washed twice in PBS and 200 μl of borate solution was added. Lysis of target cells was evaluated by measuring the calcium released into the medium after lysis with borate solution (Fig. 37).

Spontaneous release was measured in wells containing only target cells and effector cells (without antibodies). Maximum release was determined by wells containing only target cells and 1% Triton X-100. The percentage of specific antigen-mediated lysis was calculated as follows: ((x-SR) / MR-SR) * 100, where x is the average Vmax for a given concentration of antibodies, SR is the average Vmax of spontaneous release, MR is the average Vmax of maximum release.

EXAMPLE 7

The use of glyco-modified anti-EGFR monoclonal antibodies in the treatment of psoriasis.

Patients (people) suffering from psoriasis can be treated with glyco-modified anti-EGFR monoclonal antibodies obtained according to the methods of this invention. In particular, the patient receives a shock dose (400 mg / m ² ) of glyco-modified antibodies weekly by infusion. Maintenance doses of 250 mg / m ² are prescribed weekly until a complete response is achieved.

EXAMPLE 8

The use of glyco-modified anti-ErbB2 monoclonal antibodies in the treatment of metastatic prostate cancer, metastatic breast cancer, metastatic cancer of the rectum and colon, non-small cell lung cancer (stages IIIb or IV).

RhuMAb2C4 is a full-sized humanized monoclonal antibody (produced by CHO cells) directed towards ErbB2. RhuMAb2C4 blocks the binding of ErbB2 to other members of the ErbB family, thereby inhibiting intracellular signaling along the pathway associated with ErbB. RhuMAb2C4 not only inhibits the growth of tumors in which ErbB2 expression is increased, but also blocks the growth of tumors that require ErbB2 ligand-dependent signaling.

The glyco-modified form of RhuMAb2C4, created according to the methods of the present invention, can be used for monotherapy of patients with hormone-refractory (androgen-independent) cancer of the prostate, patients with metastatic breast cancer, patients with metastatic cancer of the rectum and colon, patients with non-small cell lung cancer in the stage IIIb or IV. In particular, glyco-modified RhuMAb2C4 is prescribed for intravenous administration (i.v.) weekly or once every three weeks at a dose of 2 or 4 mg / kg, respectively, until the cessation of disease progression. The antibody is supplied in the form of a multi-dose liquid dosage form (doses of 20 ml with an antibody concentration of 20 mg / ml or higher).

It should be apparent that the invention may be practiced by methods other than those described in detail in the foregoing description and examples. In light of the above ideas, numerous modifications and variations of the present invention are possible, which, therefore, are within the scope of the attached claims.

The full disclosure of all publications cited herein (including patents, patent applications, journal articles, laboratory manuals, books, and other documents) is hereby incorporated by reference.

Claims (28)

1. An isolated nucleic acid encoding a fusion polypeptide having beta-1,4-N-acetylglucosaminyl transferase III (GnTIII) or beta-1,4-galactosyltransferase (GalT) activity, said fusion polypeptide containing a mammalian or GalT catalytic GnTIII domain responsible for the localization in the Golgi complex domain of beta-1,2-N-acetylglucosaminyl transferase I (GnTI) of a mammal or mannosidase II of a mammal (Man II), wherein said mammalian GnTIII catalytic domain contains the amino acid sequence of SEQ ID NO: 21, wherein said mammalian GalT catalytic domain comprises the amino acid sequence of SEQ ID NO: 22, moreover, the specified responsible for the localization in the Golgi complex domain of the mammalian Man II contains the amino acid sequence of SEQ ID NO: 23, and and the specified localization in the Golgi complex domain of the mammalian GnTI contains the amino acid sequence of SEQ ID NO: 24. 2. The isolated nucleic acid according to claim 1, characterized in that said fusion polypeptide has GnTIII activity and contains a mammalian GnTIII catalytic domain and is responsible for the localization of the mammalian Man II domain in the Golgi complex. 3. The selected nucleic acid according to claim 2, characterized in that said catalytic domain of a mammalian GnTIII contains the amino acid sequence of SEQ ID NO: 21, and wherein said mammalian Man II domain responsible for localization in the Golgi complex contains the amino acid sequence of SEQ ID NO: 23 . 4. The selected nucleic acid according to claim 3, characterized in that said fusion polypeptide has the amino acid sequence of SEQ ID NO: 26. 5. The isolated nucleic acid according to claim 1, characterized in that said fusion polypeptide has GnTIII activity and contains the mammalian GnTIII catalytic domain and is responsible for the localization of the mammalian GnTI domain in the Golgi complex. 6. The isolated nucleic acid according to claim 5, characterized in that said catalytic domain of a mammalian GnTIII contains the amino acid sequence of SEQ ID NO: 21, while said mammalian GnTI domain responsible for localization in the Golgi complex contains the amino acid sequence of SEQ ID NO: 24. 7. The isolated nucleic acid according to claim 6, characterized in that said fusion polypeptide has the amino acid sequence of SEQ ID NO: 28. 8. The isolated nucleic acid according to claim 1, characterized in that said fusion polypeptide has GalT activity and contains a mammalian GalT catalytic domain and is responsible for the localization of the mammalian Man II domain in the Golgi complex. 9. The isolated nucleic acid according to claim 8, characterized in that said mammalian GalT catalytic domain contains the amino acid sequence of SEQ ID NO: 22, while said mammalian Man II domain responsible for localization in the Golgi complex contains the amino acid sequence of SEQ ID NO: 23 . 10. The isolated nucleic acid according to claim 1, characterized in that said fusion polypeptide has GalT activity and contains a mammalian GalT catalytic domain and is responsible for the localization of the mammalian GnTI domain in the Golgi complex. 11. The isolated nucleic acid according to claim 10, characterized in that said mammalian GalT catalytic domain contains the amino acid sequence of SEQ ID NO: 22, and wherein said mammalian GnTI domain responsible for localization in the Golgi complex contains the amino acid sequence of SEQ ID NO: 24. 12. A mammalian host cell for producing a fusion polypeptide having GnTIII or GalT activity, said mammalian host cell containing an isolated nucleic acid according to any one of claims 1 to 4. 1-11, and wherein said mammalian host cell is not a human embryonic cell. 13. A mammalian expression vector containing the isolated nucleic acid according to any one of claims. 1-11. 14. A mammalian host cell to produce a fusion polypeptide having GnTIII or GalT activity, said mammalian host cell comprising a mammalian expression vector according to claim 13, wherein said mammalian host cell is not a human embryonic cell. 15. A method for modifying the glycosylation profile of a polypeptide produced by a mammalian host cell, said method comprising administering to said mammalian host cell an isolated nucleic acid according to any one of claims. 1-11, wherein said polypeptide produced by said mammalian host cell is selected from the group consisting of a whole antibody molecule, a fragment of an antibody that retains a functional Fc region, and a fusion protein containing a region equivalent to the immunoglobulin Fc region, and wherein said mammalian host cell is not a human embryonic cell. 16. The method according to p. 15, characterized in that said polypeptide produced by said mammalian host cell is an IgG or a fragment thereof containing an IgG Fc region. 17. The method according to p. 15, characterized in that said polypeptide produced by said mammalian host cell is IgG1 or a fragment thereof containing the IgG1 Fc region. 18. The method of claim 15, wherein said polypeptide produced by said mammalian host cell is a fusion protein comprising a region equivalent to the Fc region of human IgG. 19. The method of claim 15, wherein said mammalian host cell is selected from the group consisting of CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, mouse P3X63 cells, mouse PER cells, cells PER.C6 and hybridoma cells. 20. A method of modifying the glycosylation profile of a polypeptide produced by a mammalian host cell, said method comprising introducing into the said mammalian host cell a mammalian expression vector according to claim 13, wherein said polypeptide produced by said mammalian host cell is selected from the group consisting of the whole antibody molecule, a fragment of an antibody that retains a functional Fc region, and a fusion protein containing a region equivalent to the immunoglobulin Fc region, and wherein A mammalian host cell is not a human embryonic cell .. 21. The method according to p. 20, characterized in that said polypeptide produced by said mammalian host cell is an IgG or a fragment thereof containing an IgG Fc region. 22. The method of claim 20, wherein said polypeptide produced by said mammalian host cell is IgG1 or a fragment thereof comprising an IgG1 Fc region. 23. The method according to p. 20, characterized in that said polypeptide produced by said mammalian host cell is a fusion protein containing a region equivalent to the Fc region of human IgG. 24. The method of claim 20, wherein said mammalian host cell is selected from the group consisting of CHO cells, BHK cells, NS0 cells, SP2 / 0 cells, YO myeloma cells, mouse myeloma PXX63 cells, and hybridoma cells.

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