WO2017134667A1

WO2017134667A1 - Methods of generating antibodies

Info

Publication number: WO2017134667A1
Application number: PCT/IL2017/050128
Authority: WO
Inventors: Inna BAR; Iris Pecker
Original assignee: Insight Biopharmaceuticals Ltd.
Priority date: 2016-02-02
Filing date: 2017-02-02
Publication date: 2017-08-10

Abstract

A method of altering a property of a recombinant antibody is disclosed. The method comprises: (a) analyzing the property of the antibody; (b) selecting an antibody having a property which is significantly different to a reference value; and (c) culturing cells which express the recombinant antibody selected in step (b) in a first culture medium comprising a first concentration of nicotinamide, the first concentration being between 0.5 m M - 10 m M nicotinamide, thereby altering the property of the antibody, wherein the property is selected from the group consisting of ADCC activity, glycan profile and FcγR binding.

Description

METHODS OF GENERATING ANTIBODIES

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to a method of generating antibodies, more particularly, but not exclusively, to recombinant antibodies such as recombinant anti-TNF antibodies.

Recombinant therapeutic antibodies play an important role in treatment of a large variety of diseases. Currently, more than 40 recombinant antibodies have been approved by the FDA and EMA.

Antibodies contain a target antigen- specific region which is composed of the variable regions of both the heavy and the light chains. This part of the antibody may bind and neutralize a soluble antigen or a membrane-bound target.

The Fc portion is responsible for induction of different immune system effector functions through interactions with Fc gamma receptors, Clq and neonatal receptor FcRn. These interactions induce immune system responses such as antibody dependant cell-mediated cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) and affecting monoclonal antibody (mAb) half life by binding to the neonatal receptor FcRn. Those effector functions are mediated through interaction of the effector molecules with the hinge and CH2 regions of the Fc. The CH2 domain contains an oligosaccharide located on the N-glycosylation site at position 297 of the antibody which is known to play an important role in binding to effector cells. The oligosaccharide is usually composed of a complex diantennary type with considerable heterogeneity, such as a core heptasaccharide together with additional variable outer sugar residues.

ADCC is one of the critical killing mechanisms for antibodies that bind ligands on target cells' membrane. The Fc gamma receptor (FcyR) expressed on leucocytes bind the CH2 domain of the antibodies and upon binding and creation of immune complexes with antigens on the target cells activation of the leucocytes is initiated. The activation may include phagocytosis and release of cell mediators that lead to cell permeabilization and death. The ADCC activity is dependent on the IgG isotype on the one hand, and on a specific FcyR, on the other hand. Whereas IgGl and IgG3 may induce this activity, IgG4 does not. The main FcyR that binds the IgG and is important for ADCC mechanism activation is known as the FcyRIIIa and is expressed on natural killer (NK) cells and macrophages. In many cases the ADCC activity obtained upon binding of the NK cell to the target cell is not efficient enough to perform killing of the target cell. The reason is that the affinity of the FcyRIIIa to the IgGl is low.

The interaction between FcyR and CH2 is mediated by glycosylation of the Fc domain, and is sensitive to changes in the oligosaccharide structure. The absence of core fucose in IgG results in higher binding affinity to the FcyRIIIa receptor, thereby improving (>50-fold) ADCC activity (Ferrara C, et al. J Biol Chem 2006 24;281(8):5032-6.). Therefore afucosylated antibodies are typically more potent in mediating ADCC, compared to fucose containing antibodies.

Many methods are known for generating antibodies with low fucose levels. For example, one way for obtaining antibodies with low fucose levels is to harness cells with such natural capabilities, such as Rat hybridoma YB2/0 cells (Shinkawa, Nakamura et al. 2003), although recombinant proteins produced in these cells have variable levels of fucose content. Several other possibilities of non-mammalian cells include avian cells from Vivalis, engineered aquatic plant Lemna from Biolex (Cox, Sterling et al. 2006) and variant of the moss Physocmirtella patens from Igeneon (Nechansky, Schuster et al. 2007). In addition, GlycoFi generated various lines of Pichia Pastoris cells with capabilities for several glycosylation solutions including enhanced ADCC (Hamilton, Davidson et al. 2006). In addition several mammalian cells are used for production of antibodies with various glycosylation solutions in general and enhanced ADCC in particular. Glycotope created various human glycoengineered cell lines to glyco-optimize bio-therapeutics glycosylation. Glycart, acquired by Roche, engineered a cell line producing recombinant antibodies with reduced fucose level by introducing beta(l,4)-N-acetylglucosaminyltransferase III (GnTIII), a glycosyltransferase catalyzing formation of bisected oligosaccharides that have been implicated in antibody-dependent cellular cytotoxicity (ADCC) (Umana, Jean-Mairet et al. 1999). Biowa generated a knockout in the fucosyl transferase 8 (Fut8) gene of CHO DG44 in order to diminish the fucose levels (Yamane-Ohnuki, Kinoshita et al. 2004).

Additional background art includes US Patent No. 5,691,202 and WO

2013/158275. SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided a method of altering a property of a recombinant antibody comprising:

(a) analyzing the property of the antibody;

(b) selecting an antibody having a property which is significantly different to a reference value; and

(c) culturing cells which express the recombinant antibody selected in step (b) in a first culture medium comprising a first concentration of nicotinamide, the first concentration being between 0.5 mM - 10 mM nicotinamide, thereby altering the property of the antibody, wherein the property is selected from the group consisting of ADCC activity, glycan profile and Fc gamma receptor (FcyR) binding.

According to an aspect of some embodiments of the present invention, there is provided an antibody generated according to any one of the claims 21-30, having lower amounts of GO glycoform as compared with an antibody having been produced in cells which were not cultured in media comprising L-fucose at a concentration greater than 1 mM L-fucose.

According to an aspect of some embodiments of the present invention, there is provided a cell culture medium comprising at least 0.5 mM nicotinamide and between 1- 10 mM L-fucose.

According to an aspect of some embodiments of the present invention, there is provided a method of generating an antibody comprising:

(a) culturing cells which express a recombinant antibody in a first culture medium comprising at least 0.5 mM nicotinamide;

(b) isolating the antibody; and subsequently; and

(c) testing the amount of fucosylated and/or afucosylated glycoform present in the isolated antibody, thereby generating the antibody. According to an aspect of some embodiments of the present invention there is provided a method of generating an antibody comprising culturing cells which express a recombinant antibody in a first culture medium comprising at least 1 mM L-fucose, thereby generating the antibody.

According to some embodiments of the invention, the method further comprises isolating the antibody. According to some embodiments of the invention, the method further comprises testing the amount of fucosylated and/or afucosylated glycoform present in the isolated antibody.

According to some embodiments of the invention, the method further comprises culturing cells which express the recombinant antibody in a second culture medium comprising a second concentration of nicotinamide, the second concentration being non- identical to the first concentration of nicotinamide, the second concentration being between 0.5 mM - 10 mM nicotinamide.

According to some embodiments of the invention, the activity comprises an ADCC activity.

According to some embodiments of the invention, the afucosylated glycoform is GO or Man5.

According to some embodiments of the invention, the first culture medium comprises between 3-6 mM nicotinamide.

According to some embodiments of the invention, the cells are cultured for at least two days in a second culture medium which comprises less than 0.5 mM nicotinamide prior to the culturing.

According to some embodiments of the invention, the culturing is effected for at least 6 days.

According to some embodiments of the invention, the cells comprise CHO cells.

According to some embodiments of the invention, the antibody is a monoclonal Ab (mAb).

According to some embodiments of the invention, the antibody is an antibody which binds tumor necrosis factor (TNF).

According to some embodiments of the invention, the heavy chain of the antibody has an amino acid sequence as set forth in SEQ ID NO: 1 and the light chain of the antibody has the amino acid sequence as set forth in SEQ ID NO: 2.

According to some embodiments of the invention, the method further comprises testing the activity of the antibody following the isolating.

According to some embodiments of the invention, the afucosylated glycoform is

GO or Man5. According to some embodiments of the invention, the first culture medium further comprises L-fucose.

According to some embodiments of the invention, a concentration of L-fucose is between 1-10 mM.

According to some embodiments of the invention, the concentration of L-fucose is about 6 mM.

According to some embodiments of the invention, the second culture medium is devoid of L-fucose.

According to some embodiments of the invention, the first culture medium comprises between 1-10 mM L-fucose.

According to some embodiments of the invention, the first culture medium comprises about 6 mM L-fucose.

According to some embodiments of the invention, the cells are cultured for at least two days in a second culture medium which is devoid of L-fucose prior to the culturing.

According to some embodiments of the invention, the cells are CHO cells.

According to some embodiments of the invention, the antibody binds tumor necrosis factor (TNF).

According to some embodiments of the invention, the method further comprises isolating the antibody.

According to some embodiments of the invention, the method further comprises testing the amount of GO glycoform present in the antibody. According to some embodiments of the invention, the antibody has a heavy chain amino acid sequence as set forth in SEQ ID NO: 1 and a light chain amino acid sequence as set forth in SEQ ID NO: 2.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

Figure 1 is a bar graph demonstrating the effect of nicotinamide addition to the culture media on day 0 or 3 on the percentage of mAbl8 GO glycoform, as evaluated by LabChip GXII. Nicotinamide was added to the culture media at the indicated concentrations.

Figure 2 is a bar graph demonstrating the effect of nicotinamide addition to the culture media on day 0 or 3 on the percentage of mAbl8 binding to FcyRIIIa, as evaluated by Octet. Nicotinamide was added to the culture media at the indicated concentrations.

Figure 3 is a graph demonstrating the correlation between mAbl8 ADCC activity and GO content. Nicotinamide was added to the culture media on day 0 or day 3 at the indicated concentrations and mAbl8 ADCC activity and the percentage of GO glycoform were plotted on a dual axis graph. DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to a method of generating antibodies, more particularly, but not exclusively, to recombinant antibodies, such as anti-TNF antibodies.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

It is essential that biosimilar medicines have a primary structure identical to their reference products (e.g., amino acid sequences must be identical) and also show similar activity. In the case of monoclonal antibodies (mAbs), due to their more complex structure, a greater level of demand is in order and identity at other levels (e.g., post- translational modifications including the level of fucosylation within the Fc region of the molecule) have to be shown to establish "similarity". Furthermore, FcyRIIIa binding and ADCC activity must also be similar.

The present inventors have now discovered a way to control the level of fucosylation of antibodies paving the way for the generation of antibodies (e.g. monoclonals or Fc fusion proteins) having a greater degree of biosimilarity to their reference products.

Specifically, the present inventors have uncovered that culturing cells which are genetically modified to express recombinant antibodies in a medium comprising nicotinamide or L-fucose, reduces the amount of afucosylated glycoforms of the antibody. Furthermore, the present inventors showed that this effect was both concentration-dependent and time-dependent.

As is illustrated hereunder and in the examples section, which follows, the present inventors show that culturing of CHO cells which are genetically modified to express an anti-TNF monoclonal antibody in a medium comprising about 0.5-10 mM nicotinamide or about 1-10 mM L-fucose, reduced the level of at least one afucosylated glycoform (e.g. GO) of the antibody.

Whilst further reducing the present invention to practice, the present inventors showed that the level of GO glycoform correlated with FcyRIIIa binding and ADCC activity. Consequently, the present teachings suggest that the level of GO may be used as a proxy for ADCC activity and it can be controlled by culturing antibody expressing cells in media containing nicotinamide or L-fucose.

Thus, according to a first aspect of the present invention, there is provided a method of generating an antibody comprising culturing cells which express a recombinant antibody in a first culture medium comprising at least 0.5 mM nicotinamide, thereby generating the antibody.

According to another aspect of the present invention, there is provided a method of generating an antibody comprising culturing cells which express a recombinant antibody in a first culture medium comprising at least 1 mM L-fucose, thereby generating the antibody.

The term "antibody" refers to an immunoglobulin molecule which comprises four polypeptide chains, two heavy (H) chains and two light (L) chains inter- connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region (CH). The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy- terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

According to one embodiment, the antibody is not an antibody fragment comprised solely of the antigen binding portion, but also comprises an Fc region. Thus, for example according to this embodiment, the antibody does not consist solely of (i) a Fab fragment, a monovalent fragment comprising the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment comprising the VH and CHI domains; (iv) a Fv fragment comprising the VL and VH domains of a single arm of an antibody, (v) a dAb fragment which comprises a VH domain; or (vi) an isolated complementarity determining region (CDR). In another embodiment, the antibody is an Fc fusion protein.

The antibody may be monospecific, (i.e. recognize a single antigen) or bispecific (each arm of the antibody recognizing a different antigen).

The antibody may be of any class e.g. IgAi, IgA₂, IgD, IgE, IgGi, IgG₂, IgG₃, IgG₄, and IgM antibodies.

Preferably the antibody is of the class IgG - e.g. IgGiK.

The antibody may be a natural human antibody, a humanized and human-type antibody prepared by genetic recombination, a monoclonal antibody of mice. Humanized and human-type monoclonal antibodies are the most useful from an industrial perspective.

Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a CDR of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(l):86-95 (1991)]. Similarly, human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10,: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).

In one embodiment, the antibody of the invention binds to TNFa - e.g. human TNFa. The phrase "Tumor necrosis factor-a" (abbreviated herein as hTNFa or TNFa) is a multifunctional pro-inflammatory cytokine secreted predominantly by monocytes/macrophages that has effects on lipid metabolism, coagulation, insulin resistance, and endothelial function. TNFa is a soluble homotrimer of 17 kD protein subunits. A membrane-bound 26 kD precursor form of TNFa also exists. It is found in synovial cells and macrophages in tissues. Cells other than monocytes or macrophages also produce TNFa. For example, human non-monocytic tumor cell lines produce TNFa as well as CD4+ and CD8+ peripheral blood T lymphocytes and some cultured T and B cell lines produce TNFa. The nucleic acid encoding TNFa is available as GenBank Accession No. X02910 and the polypeptide sequence is available as GenBank Accession No. CAA26669. The term human TNFa is intended to include recombinant human TNFa (rh TNFa), which can be prepared by standard recombinant expression methods.

Examples of anti-TNFa antibodies include, but are not limited to, anti-TNFa human antibodies as well as those described in U.S. Patent Nos. 6,090,382; 6,258,562; 6,509,015, and in U.S. Patent Application Serial Nos. 09/801185 and 10/302356, each of which is incorporated by reference herein. In one embodiment, the TNFa antibody is infliximab (Remicade^®, Johnson and Johnson; described in U.S. Patent No. 5,656,272, incorporated by reference herein), CDP571 (a humanized monoclonal anti-TNF- alpha IgG4 antibody), CDP 870 (a humanized monoclonal anti-TNF-alpha antibody fragment), an anti-TNF dAb (Peptech), CNTO 148 (golimumab; Medarex and Centocor), antibodies described in WO 02/12502, and adalimumab (Humira^® Abbott Laboratories, a human anti-TNF Ab, described in US 6,090,382 as D2E7).

As used herein, the term "adalimumab," refers to a FDA approved fully humanized IgGl, TNF-alpha inhibitor monoclonal antibody (tradename Humira®) produced by Abbott Laboratories. Each IgG antibody molecule comprises two kappa light chains and two human IgGl heavy chains, the total molecular weight of Adalimumab is 148 kDa. Each light chain consists of 214 amino acid residues and each heavy chain consists of 451 amino acid residues.

It will be appreciated that the antibody may be a biosimilar of the above mentioned antibodies.

As used herein, the term "biosimilar" refers to a biopharmaceutical which is deemed to be comparable in quality, safety, and efficacy to reference product marketed by an innovator company.

The invention also contemplates antibodies which have sequences at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, identical or homologous to those disclosed herein for the particular antibodies described herein above. When the antibodies are not 100 % identical to those described herein above, it is conceived that they may comprise either conservative or non-conservative amino acid changes.

The phrase "conservative variation" as used herein refers to the replacement of an amino acid residue by another, biologically similar residue. Examples of conservative variations include the substitution of one hydrophobic residue such as isoleucine, valine, leucine, or methionine for another, or the substitution of one solar residue for another, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, and the like. The term "conservative variation" also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide.

In a particular embodiment, the antibody is one which is known to have the same amino acid sequence as a reference product marketed by a company, but has an enhanced activity as compared to the reference product.

Thus, according to a particular embodiment, prior to culturing cells which express the recombinant antibody in the nicotinamide (or fucose) as detailed herein, the activity (or a particular property) of the antibody is tested. If the antibody has an activity above a predetermined level (e.g. at least 1 %, 2%, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 15 %, 20 %, 25 %, 30 %, 40 %, 50 %, 60 % 70 %, 80 % 90 % 100% higher than a reference antibody having the same amino acid sequence), then the cells which express the recombinant antibody are cultured in the presence of nicotinamide (e.g. between 0.5 mM - 10 mM nicotinamide) or fucose. Alternatively, if the antibody has an amount of afucosylated glycans above a predetermined level (e.g. at least 1 %, 2%, 3 %, 4 %, 5 %, 6 %, 7 %, 8 %, 9 %, 10 %, 15 %, 20 %, 25 %, 30 %, 40 %, 50 %, 60 % 70 %, 80 % 90 % 100% higher than a reference antibody having the same amino acid sequence), then the cells which express the recombinant antibody are cultured in the presence of nicotinamide (e.g. between 0.5 mM - 10 mM nicotinamide) or fucose. The present invention further contemplates analyzing various concentrations of nicotinamide and/or fucose (e.g. between 0.5 mM - 10 mM nicotinamide) so as to select the optimum concentration for a particular clone so as to bring the activity as close as possible to the reference antibody or to the desired level. Exemplary methods for analyzing antibody activity include for example analyzing binding activity to its target antigen, FcyRIIIa and FcyRIIIb binding activity and ADCC activity, as described in the Examples section herein below.

In addition to (or instead of) analyzing activity, the present inventors further contemplate analyzing the glycan content of the antibody and in particular testing the amount of afucosylated glycoforms (e.g. GO glycoform, Man5 glycoform, G- glycoform, GO-GN glycoform, Gl glycoform, G' l glycoform or G2 glycoform) present in the antibody. This may be carried out by capillary electrophoresis or other methods described in the art.

According to a particular embodiment, the antibody is a recombinant, monoclonal antibody.

Methods of producing polyclonal and monoclonal antibodies are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).

Antibodies may be produced by immunization of a non-human animal, preferably a mouse, with an immunogen comprising a desired antigen or immunogen. Alternatively, antibodies may be provided by selection of combinatorial libraries of immunoglobulins, as disclosed for instance in Ward et al (Nature 341 (1989) 544). According to a particular embodiment, the antibody is generated in vitro (i.e. not by injecting the antigen into a living animal).

The step of immunizing a non-human mammal with an antigen may be carried out in any manner well known in the art for stimulating the production of antibodies in a mouse (see, for example, E. Harlow and D. Lane, Antibodies: A Laboratory Manual., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988)). In a preferred embodiment, the non-human animal is a mammal, such as a rodent (e.g., mouse, rat, etc.), bovine, porcine, horse, rabbit, goat, sheep, etc. As mentioned, the non-human mammal may be genetically modified or engineered to produce "human" antibodies. Typically, the immunogen is suspended or dissolved in a buffer, optionally with an adjuvant, such as complete Freund's adjuvant. Methods for determining the amount of immunogen, types of buffers and amounts of adjuvant are well known to those of skill in the art and are not limiting in any way on the present invention. These parameters may be different for different immunogens, but are easily elucidated. Similarly, the location and frequency of immunization sufficient to stimulate the production of antibodies is also well known in the art. In a typical immunization protocol, the non-human animals are injected intraperitoneally with antigen on day 1 and again about a week later. This is followed by recall injections of the antigen around day 20, optionally with adjuvant such as incomplete Freund's adjuvant. The recall injections are performed intravenously or intraperitoneally and may be repeated for several consecutive days. This is followed by a booster injection at day 40, either intravenously or intraperitoneally, typically without adjuvant. This protocol results in the production of antigen-specific antibody-producing B cells after about 40 days. Other protocols may also be utilized as long as they result in the production of B cells expressing an antibody directed to the antigen used in immunization.

In an alternate embodiment, lymphocytes from a non-immunized non-human mammal are isolated, grown in vitro, and then exposed to the immunogen in cell culture. The lymphocytes are then harvested and the fusion step described below is carried out.

For monoclonal antibodies, the next step is the isolation of splenocytes from the immunized non-human mammal and the subsequent fusion of those splenocytes with an immortalized cell in order to form an antibody-producing hybridoma. The isolation of splenocytes from a non-human mammal is well-known in the art and typically involves removing the spleen from an anesthetized non-human mammal, cutting it into small pieces and squeezing the splenocytes from the splenic capsule and through a nylon mesh of a cell strainer into an appropriate buffer so as to produce a single cell suspension. The cells are washed, centrifuged and re-suspended in a buffer that lyses any red blood cells. The solution is again centrifuged and remaining lymphocytes in the pellet are finally re-suspended in fresh buffer.

Once isolated and present in single cell suspension, the lymphocytes are fused to an immortal cell line. This is typically a mouse myeloma cell line, although many other immortal cell lines useful for creating hybridomas are known in the art.

Preferred murine myeloma lines include, but are not limited to, those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. U.S.A., X63 Ag8653 and SP-2 cells available from the American Type Culture Collection, Rockville, Md. U.S.A. The fusion is effected using polyethylene glycol or the like. The resulting hybridomas are then grown in selective media that contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

The hybridomas are typically grown on a feeder layer of macrophages. The macrophages are preferably from littermates of the non-human mammal used to isolate splenocytes and are typically primed with incomplete Freund's adjuvant or the like several days before plating the hybridomas. Fusion methods are described in (Goding, "Monoclonal Antibodies: Principles and Practice," pp. 59-103, Academic Press, 1986).

The cells are allowed to grow in the selection media for sufficient time for colony formation and antibody production. This is usually between 7 and 14 days. The hybridoma colonies are then assayed for the production of antibodies that bind the immunogen/antigen. The assay is typically a colorimetric ELISA-type assay, although any assay may be employed that can be adapted to the wells that the hybridomas are grown in. Other assays include immunoprecipitation and radioimmunoassay. The wells positive for the desired antibody production are examined to determine if one or more distinct colonies are present. If more than one colony is present, the cells may be re- cloned and grown to ensure that only a single cell has given rise to the colony producing the desired antibody. Positive wells with a single apparent colony are typically recloned and re-assayed to insure only one monoclonal antibody is being detected and produced.

Hybridomas that are confirmed to be producing a monoclonal antibody are then grown up in larger amounts in an appropriate medium, such as DMEM or RPMI-1640. Alternatively, the hybridoma cells can be grown in vivo as ascites tumors in an animal.

After sufficient growth to produce the desired monoclonal antibody, the growth media containing monoclonal antibody (or the ascites fluid) is separated away from the cells and the monoclonal antibody present therein is purified. DNA encoding the heavy and light chains of the antibody may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of antibodies such as murine or human). Once isolated, the DNA can be ligated into expression vectors, which are then transfected into host cells.

As mentioned, the antibodies according to the invention are typically produced by recombinant means.

To express a recombinant antibody of the invention, DNAs encoding partial or full-length light and heavy chains are inserted into one or more expression vector such that the genes are operatively linked to transcriptional and translational control sequences. (See, e.g., U.S. Pat. No. 6,914,128, the entire teaching of which is incorporated herein by reference). In this context, the term "operatively linked" is intended to mean that an antibody gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into a separate vector or, more typically, both genes are inserted into the same expression vector. The antibody genes are inserted into an expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or blunt end ligation if no restriction sites are present). Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non- immunoglobulin protein). In addition to the antibody chain genes, a recombinant expression vector of the invention can carry one or more regulatory sequence that controls the expression of the antibody chain genes in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such regulatory sequences are described, e.g., in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990), the entire teaching of which is incorporated herein by reference. It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Suitable regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma. For further description of viral regulatory elements, and sequences thereof, see, e.g., U.S. Patent No. 5,168,062 by Stinski, U.S. Patent No. 4,510,245 by Bell et al. and U.S. Patent No. 4,968,615 by Schaffher et al., the entire teachings of which are incorporated herein by reference. In addition to the antibody chain genes and regulatory sequences, a recombinant expression vector of the invention may carry one or more additional sequences, such as a sequence that regulates replication of the vector in host cells (e.g., origins of replication) and/or a selectable marker gene. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Patents Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al, the entire teachings of which are incorporated herein by reference). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, on a host cell into which the vector has been introduced. Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr- host cells with methotrexate selection/amplification) and the neo gene (for G418 selection).

To express an antibody recombinantly, a host cell is transfected with one or more recombinant expression vector carrying DNA fragments encoding the immunoglobulin light and heavy chains of the antibody such that the light and heavy chains are expressed in the host cell and secreted into the medium in which the host cells are cultured, from which medium the antibodies can be recovered. Standard recombinant DNA methodologies are used to obtain antibody heavy and light chain genes, incorporate these genes into recombinant expression vectors and introduce the vectors into host cells, such as those described in Sambrook, Fritsch and Maniatis (eds), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N. Y., (1989), Ausubel et al. (eds.) Current Protocols in Molecular Biology, Greene Publishing Associates, (1989) and in U.S. Patent Nos. 4,816,397 & 6,914,128, the entire teachings of which are incorporated herein.

For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains is (are) transfected into a host cell by standard techniques. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into aprokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE- dextran transfection and the like. Although it is theoretically possible to express the antibodies of the invention in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells, such as mammalian host cells, is suitable because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody.

Suitable host cells for the expression of glycosylated antibodies are those derived from multicellular organisms. Examples of invertebrate cells of multicellular organisms include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fraitfly), and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-I variant of Autographa calif ornica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells. Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco can also be utilized as hosts.

Suitable mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary (CHO cells) (including dhfr- CHO cells, described in Urlaub and Chasin, (1980) PNAS USA 77:4216-4220, used with a DHFR selectable marker, e.g., as described in Kaufman and Sharp (1982) Mol. Biol. 159:601- 621, the entire teachings of which are incorporated herein by reference), NSO myeloma cells, COS cells and SP2 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown. Other examples of useful mammalian host cell lines are monkey kidney CVl line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CVl ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (Wl 38, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N. Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2), the entire teachings of which are incorporated herein by reference.

Host cells are transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

As mentioned, once cells which express a recombinant antibody are obtained and it is required to reduce the level of afucosylated glycoforms, they are cultured in a medium comprising at least 0.5 mM nicotinamide and/or at least 1 mM L-fucose.

Commercially available media such as Ham's F 10™ (Sigma), Minimal Essential Medium™ ((MEM), (Sigma), RPMI- 1640 (Sigma), and Dulbecco's Modified Eagle's Medium™ ((DMEM), Sigma) are suitable for culturing the host cells, according to this aspect of the present invention. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. No. Re. 30,985 may be used as culture media for the host cells, the entire teachings of which are incorporated herein by reference.

Nicotinamide (NA), also known as "niacinamide", is the amide derivative form of Vitamin B3 (niacin) which is thought to preserve and improve beta cell function. Nicotinamide has the chemical formula C6¾N₂0. Nicotinamide is essential for growth and the conversion of foods to energy, and it has been used in arthritis treatment and diabetes treatment and prevention.

Nicotinamide (NA)

According to a particular embodiment, the nicotinamide is a nicotinamide derivative or a nicotinamide mimic. The term "derivative of nicotinamide (NA)" as used herein denotes a compound which is a chemically modified derivative of the natural NA. In one embodiment, the chemical modification may be a substitution of the pyridine ring of the basic NA structure (via the carbon or nitrogen member of the ring), via the nitrogen or the oxygen atoms of the amide moiety. When substituted, one or more hydrogen atoms may be replaced by a substituent and/or a substituent may be attached to a N atom to form a tetravalent positively charged nitrogen. Thus, the nicotinamide of the present invention includes a substituted or non-substituted nicotinamide. In another embodiment, the chemical modification may be a deletion or replacement of a single group, e.g. to form a thiobenzamide analog of NA, all of which being as appreciated by those versed in organic chemistry. The derivative in the context of the invention also includes the nucleoside derivative of NA (e.g. nicotinamide adenine). A variety of derivatives of NA are described, some also in connection with an inhibitory activity of the PDE4 enzyme (WO03/068233; WO02/060875; GB2327675A), or as VEGF-receptor tyrosine kinase inhibitors (WO01/55114). For example, the process of preparing 4-aryl-nicotinamide derivatives (WO05/014549). Other exemplary nicotinamide derivatives are disclosed in WO01/55114 and EP2128244.

The concentration of nicotinamide according to this aspect of the present invention is typically above 0.5 mM. In one embodiment, the concentration of nicotinamide is between 0.5 - 10 mM. In one embodiment, the concentration of nicotinamide is between 2.5 - 10 mM. In another embodiment, the concentration of nicotinamide is between 3-8 mM, more preferably between 3-6 mM. In another embodiment, the concentration of nicotinamide is between 4-6 mM.

The cells are cultured with at least 0.5 mM nicotinamide for at least 6 days e.g. between 7-13 days.

Optionally, prior to culturing the antibody-expressing cells with nicotinamide, the cells are cultured with a medium which comprises less than 0.5 mM nicotinamide. Preferably, this culturing step is carried out for no more than 10 days, more preferably no more than 8 days, more preferably no more than 6 days, more preferably no more than 4 days, no more than 3 days or no more than 2 days. At a minimum, this step is effected for at least 1 day.

The medium which is used for this pre-culturing phase is preferably the same medium which is used for the culturing stage in the presence of at least 0.5 mM nicotinamide. Such media have been described herein above.

L-fucose (6-deoxy-L-galactose) is a monosaccharide that is a common component of many N- and O-linked glycans and glycolipids produced by mammalian cells. Two structural features distinguish fucose from other six-carbon sugars. These include the lack of a hydroxyl group on the carbon at the 6-position (C-6) and the L- configuration.

L-fucose is commercially available from Sigma (Cat#F2252).

Contemplated concentrations of L-fucose are between 1-10 mM, more preferably between 2-8 mM, more preferably between 3-7 mM - e.g. about 6 mM.

The cells are cultured with at least 1 mM L-fucose for at least 6 days, for example 6-15 days.

Optionally, prior to culturing the antibody- expressing cells with L-fucose, the cells are cultured with a medium which is devoid of L-fucose (or at least contains less than 0.1 mM L-fucose). Preferably, this culturing step is effected for no more than 10 days, more preferably no more than 8 days, more preferably no more than 6 days, more preferably no more than 4 days, no more than 3 days or no more than 2 days. At a minimum, this step is effected for at least 1 day.

The medium which is used for this pre-culturing phase is preferably the same medium which is used for the culturing stage in the presence of at least 1 mM L-fucose. Such media have been described herein above. In a particular embodiment, when the cells are cultured in at least 2 mM nicotinamide, L-fucose is present in the medium at a concentration of at least 1 mM, (for example about 6 mM L-fucose).

Thus, according to another aspect of the present invention there is provided a cell culture medium comprising at least 0.5 mM nicotinamide and between 1-10 mM L- fucose. (For example a cell culture medium comprising between 2-4 mM nicotinamide and 6 mM L-fucose is contemplated).

According to still another aspect there is provided a cell culture comprising mammalian cells which express an antibody and a culture medium comprising at least 0.5 mM nicotinamide, and more preferably between 2-6 mM nicotinamide.

The cell culture may further comprise L-fucose (e.g. at concentrations of at least 1 mM, (e.g. between 1-10 mM and more preferably between about 2-6 mM), as further detailed herein above).

Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as gentamycin drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

Following culturing of the cells which express the recombinant antibody, the antibody may then be isolated.

Depending on the cell type, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed cells (e.g., resulting from homogenization), can be removed, e.g., by centrifugation or ultrafiltration.

Lysis of the cells may be performed by a variety of methods, including mechanical shear, osmotic shock, or enzymatic treatments. Such disruption releases the entire contents of the cell into the homogenate, and in addition produces subcellular fragments that are difficult to remove due to their small size. These are generally removed by differential centrifugation or by filtration. Where the antibody is secreted, supernatants can be treated according to methods known in the art so as to further purify the antibody.

Exemplary antibody purification techniques include for example use of affinity resins (e.g. protein A resin), cation exchange (CEX) resin; anion exchange resins, mixed mode (MM) resins, use of a hydrophobic interaction chromatography (HIC) medium. Such methods are disclosed in WO2007/117490; WO2010/141039; WO2013/158279; WO2013/176754; WO2013/066707 and WO2011/015919.

Following the isolating, the antibody may be analyzed for activity.

Exemplary methods for analyzing antibody activity include for example analyzing binding activity to its target antigen, FcyRIIIa binding activity and ADCC activity, as described in the Examples section herein below.

In addition to (or instead of) analyzing activity, the present inventors further contemplate analyzing the glycan content of the antibody and in particular testing the amount of GO glycoform present in the antibody. This may be carried out by capillary electrophoresis or other methods described in the art.

The novel methods of culturing the cells expressing the recombinant antibodies described in the present application result in the antibodies themselves also being novel.

Thus according to another aspect of the present an antibody generated according to the methods described herein.

Typically, the antibodies have lower amounts of at least one afucosylated glycoform (e.g. GO, Gl, Gl', G2 or Man5) as compared with an antibody having been produced in cells which were not cultured in media comprising nicotinamide at a concentration greater than 0.5 mM nicotinamide. In one embodiment, the antibodies have lower amounts of GO or Man5. In other embodiments the antibodies have lower amounts of both GO and Man5.

Furthermore, the antibodies have lower amounts of GO glycoform as compared with an antibody having been produced in cells which were not cultured in media comprising L-fucose at a concentration greater than 1 mM L-fucose. In one embodiment, the antibodies have less than about 95 % of an afucosylated glycoform as compared with an antibody having been produced in cells which were not cultured in media comprising nicotinamide at a concentration greater than 0.5 mM nicotinamide.

In one embodiment, the antibodies have less than about 90 % of an afucosylated glycoform as compared with an antibody having been produced in cells which were not cultured in media comprising nicotinamide at a concentration greater than 0.5 mM nicotinamide.

In one embodiment, the antibodies have less than about 85 % of an afucosylated glycoform as compared with an antibody having been produced in cells which were not cultured in media comprising nicotinamide at a concentration greater than 0.5 mM nicotinamide.

In one embodiment, the antibodies have less than about 80 % of an afucosylated glycoform as compared with an antibody having been produced in cells which were not cultured in media comprising nicotinamide at a concentration greater than 0.5 mM nicotinamide.

In one embodiment, the antibodies have less than about 75 % of an afucosylated glycoform as compared with an antibody having been produced in cells which were not cultured in media comprising nicotinamide at a concentration greater than 0.5 mM nicotinamide.

In one embodiment, the antibodies have less than about 70 % of an afucosylated glycoform as compared with an antibody having been produced in cells which were not cultured in media comprising nicotinamide at a concentration greater than 0.5 mM nicotinamide.

In one embodiment, the antibodies have less than about 65 % of an afucosylated glycoform as compared with an antibody having been produced in cells which were not cultured in media comprising nicotinamide at a concentration greater than 0.5 mM nicotinamide.

In one embodiment, the antibodies have less than about 60 % of an afucosylated glycoform as compared with an antibody having been produced in cells which were not cultured in media comprising nicotinamide at a concentration greater than 0.5 mM nicotinamide. In one embodiment, the antibodies have less than about 55 % of an afucosylated glycoform as compared with an antibody having been produced in cells which were not cultured in media comprising nicotinamide at a concentration greater than 0.5 mM nicotinamide.

In one embodiment, the antibodies have less than about 50 % of an afucosylated glycoform as compared with an antibody having been produced in cells which were not cultured in media comprising nicotinamide at a concentration greater than 0.5 mM nicotinamide.

In one embodiment, the antibodies have less than about 95 % of GO glycoform as compared with an antibody having been produced in cells which were not cultured in media comprising L-fucose at a concentration greater than 1 mM L-fucose.

In one embodiment, the antibodies have less than about 90 % of GO glycoform as compared with an antibody having been produced in cells which were not cultured in media comprising L-fucose at a concentration greater than 1 mM L-fucose.

In one embodiment, the antibodies have less than about 85 % of GO glycoform as compared with an antibody having been produced in cells which were not cultured in media comprising L-fucose at a concentration greater than 1 mM L-fucose.

In one embodiment, the antibodies have less than about 80 % of GO glycoform as compared with an antibody having been produced in cells which were not cultured in media comprising L-fucose at a concentration greater than 1 mM L-fucose.

In one embodiment, the antibodies have less than about 75 % of GO glycoform as compared with an antibody having been produced in cells which were not cultured in media comprising L-fucose at a concentration greater than 1 mM L-fucose.

In one embodiment, the antibodies have less than about 70 % of GO glycoform as compared with an antibody having been produced in cells which were not cultured in media comprising L-fucose at a concentration greater than 1 mM L-fucose.

In one embodiment, the antibodies have less than about 65 % of GO glycoform as compared with an antibody having been produced in cells which were not cultured in media comprising L-fucose at a concentration greater than 1 mM L-fucose.

In one embodiment, the antibodies have less than about 60 % of GO glycoform as compared with an antibody having been produced in cells which were not cultured in media comprising L-fucose at a concentration greater than 1 mM L-fucose. In one embodiment, the antibodies have less than about 55 % of GO glycoform as compared with an antibody having been produced in cells which were not cultured in media comprising L-fucose at a concentration greater than 1 mM L-fucose.

In one embodiment, the antibodies have less than about 50 % of GO glycoform as compared with an antibody having been produced in cells which were not cultured in media comprising L-fucose at a concentration greater than 1 mM L-fucose.

Antibodies obtained using the process of the invention may be incorporated into pharmaceutical compositions suitable for administration to a subject. Typically, the pharmaceutical composition comprises an antibody and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Examples of pharmaceutically acceptable carriers include one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of antibody, or antigen-binding portion thereof.

Pharmaceutical compositions comprising antibodies generated using the methods of the invention may be found in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes and suppositories. The preferred form depends on the intended mode of administration and therapeutic application. Typical preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies or other TNFa inhibitors. The preferred mode of administration is parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the antibody is administered by intravenous infusion or injection. In another preferred embodiment, the antibody is administered by intramuscular or subcutaneous injection. Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, dispersion, liposome, or other ordered structure suitable to high drug concentration. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., antibody, or antigen-binding portion thereof) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.

Supplementary active compounds can also be incorporated into the compositions. In certain embodiments, an antibody, or antigen-binding portion thereof, for use in the methods of the invention is coformulated with and/or coadministered with one or more additional therapeutic agents. For example, an anti-hTNFa antibody or antibody portion of the invention may be coformulated and/or coadministered with one or more DMARD or one or more NSAID or one or more additional antibodies that bind other targets (e.g., antibodies that bind other cytokines or that bind cell surface molecules), one or more cytokines, soluble TNFa receptor (see e.g., PCT Publication No. WO 94/06476) and/or one or more chemical agents that inhibit hTNFa production or activity (such as cyclohexane-ylidene derivatives as described in PCT Publication No. WO 93/19751) or any combination thereof. Furthermore, one or more antibodies of the invention may be used in combination with two or more of the foregoing therapeutic agents. Such combination therapies may advantageously utilize lower dosages of the administered therapeutic agents, thus avoiding possible side effects, complications or low level of response by the patient associated with the various monotherapies.

In one embodiment, the invention includes pharmaceutical compositions comprising an effective amount of an anti-TNFa antibody, or antigen-binding portion thereof, and a pharmaceutically acceptable carrier, wherein the effective amount of the anti-TNFa antibody may be effective to treat a TNFa-related disorder, including, for example, Crohn's disease. In one embodiment, the antibody or antibody portion is incorporated into a pharmaceutical formulation as described in PCT/IB03/04502 and U.S. Application No. 10/222140, incorporated by reference herein. This formulation includes a concentration 50 mg/ml of the antibody adalimumab, wherein one pre-filled syringe contains 40 mg of antibody for subcutaneous injection.

The antibodies, or antibody-portions, obtained using the methods of the present invention can be administered by a variety of methods known in the art, although for many therapeutic applications, the preferred route/mode of administration is subcutaneous injection. In another embodiment, administration is via intravenous injection or infusion. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results. In certain embodiments, the active compound may be prepared with a carrier that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

The antibodies, or antigen-binding portion thereof, obtained using the methods of the invention can also be administered in the form of protein crystal formulations which include a combination of protein crystals encapsulated within a polymeric carrier to form coated particles. The coated particles of the protein crystal formulation may have a spherical morphology and be microspheres of up to 500 micro meters in diameter or they may have some other morphology and be microparticulates. The enhanced concentration of protein crystals allows the antibody of the invention to be delivered subcutaneously. In one embodiment, the antibodies of the invention are delivered via a protein delivery system, wherein one or more of a protein crystal formulation or composition, is administered to a subject with a TNF-related disorder. Compositions and methods of preparing stabilized formulations of whole antibody crystals or antibody fragment crystals are also described in WO 02/072636, which is incorporated by reference herein. In one embodiment, a formulation comprising the crystallized antibody fragments described in PCT/IB03/04502 and U.S. Application No. 10/222140, incorporated by reference herein, are used to treat a TNFa-related disorder using the multiple- variable dose methods of the invention.

In certain embodiments, an antibodies, or antigen-binding portion thereof, obtained using the methods of the invention may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound (and other ingredients, if desired) may also be enclosed in a hard or soft shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound of the invention by other than parenteral administration, it may be necessary to coat the compound with, or coadminister the compound with, a material to prevent its inactivation. The pharmaceutical compositions of the invention may include a "therapeutically effective amount" or a "prophylactically effective amount" of an antibody or antigen- binding portion thereof of the invention. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the antibody, or antigen- binding portion thereof, may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody, antibody portion, other.

A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody, or antigen-binding portion thereof, are outweighed by the therapeutically beneficial effects. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactic ally effective amount will be less than the therapeutically effective amount. Dosage regimens may be adjusted to provide the optimum desired response {e.g., a therapeutic or prophylactic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit comprising a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody, or antigen-binding portion thereof, is 10 to 200 mg, more preferably 20 to 160 mg, more preferably 40 to 80 mg, and most preferably 80 mg. In one embodiment, the therapeutically effective amount of an antibody or, antigen- binding portion thereof, is about 20 mg. In another embodiment, the therapeutically effective amount of an antibody or portion thereof is about 40 mg. In still another embodiment, the therapeutically effective amount of an antibody or, antigen-binding portion thereof, is about 80 mg. In one embodiment, the therapeutically effective amount of an antibody or portion thereof for use in the methods of the invention is about 120 mg. In yet another embodiment, the therapeutically effective amount of an antibody, or antigen -binding portion thereof, is about 160 mg. Ranges intermediate to the above recited dosages, e.g. about 78.5 to about 81.5; about 15 to about 25; about 30 to about 50; about 60 to about 100; about 90 to about 150; about 120 to about 200, are also intended to be part of this invention. For example, ranges of values using a combination of any of the above recited values as upper and/or lower limits are intended to be included. It is to be noted that dosage values may vary with the type and severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

Antibodies, or antibody-portions thereof, obtained using the methods of the invention may be administered on a biweekly dosing regimen as described in WO02/100330, a low dose regimen as described in WO 04/037205, and a multiple variable dosing regimen as described in WO 05/110452, each of which is incorporated by reference herein.

The invention also pertains to packaged pharmaceutical compositions, articles of manufacture, or kits comprising the antibody, or antigen-binding portion thereof, obtained using the process of the invention. The article of manufacture may comprise an antibody, or antigen-binging portion thereof, obtained using the method of the invention and packaging material. The article of manufacture may also comprise label or package insert indicating the formulation or composition comprising the antibody, or antigen- binding portion thereof, has a reduced level of GO glycoform. In addition, the article of manufacture may comprise a label or package insert contained within the packaging material indicating that the adalimumab formulation comprises no greater than about 70 ng/mg of host cell protein (HCP) or a label or package insert contained within the packaging material indicating that the adalimumab formulation comprises no greater than about 13 ng/mg. The article of manufacture may comprise a label or package insert contained within the packaging material indicating that the adalimumab formulation comprises no greater than about 5 ng HCP/mg adalimumab. The article of manufacture may also comprise packaging material indicating that the adalimumab formulation comprises no greater a level of procathepsin L than that indicated by a cathepsin L activity of about 3.0 RFU/s/mg adalimumab.

The antibodies generated using the methods described herein can be used for inhibiting TNFa activity in a subject suffering from a disorder in which TNFa activity is detrimental. TNFa has been implicated in the pathophysiology of a wide variety of disorders (see e.g., Moeller, A., et al. (1990) Cytokine 2: 162-169; U.S. Patent No. 5,231,024 to Moeller et al; European Patent Publication No. 260610 Bl by Moeller, A.) including sepsis, infections, malignancies, autoimmune diseases, pulmonary disorders, intestinal disorders, transplant rejection and graft- versus-host disease.

Examples of autoimmune conditions include rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis and gouty arthritis, allergy, multiple sclerosis, autoimmune diabetes, arthritis, metabolic diseases, autoimmune uveitis, lupus, Crohn's disease, cardiac disorders and nephrotic syndrome. Other examples of autoimmune conditions include multisystem autoimmune diseases and autoimmune hearing loss. A more extensive list of diseases which may be treated using the antibodies purified according to the present methods is set forth in WO2007117490, the contents of which is incorporated herein.

It is expected that during the life of a patent maturing from this application many relevant anti-TNFalpha antibodies will be developed and the scope of the term anti- TNFalpha antibody is intended to include all such new technologies a priori.

As used herein, the terms "about" or "approximately" used with a pH or pi (isoelectric point) value refers to a variance of 0.1, 0.2, 0.3, 0.4 or 0.5 units. When used with a temperature value, "about" or "approximately" refers to a variance of 1, 2, 3, 4 or 5 degrees. When used with other values, such as length and weight, "about" or "approximately" refers to a variance of 1%, 2%, 3%, 4% or 5%.

When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".

The term "consisting of means "including and limited to". The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

As used herein, the term "treating" includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley- Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

MATERIALS AND METHODS

Basal Medium - Basal media used for culture propagation and fed-batch is mix of two serum-free media. The media mix was supplemented with 4mM of L-glutamine (Lonza). During fed batch culture was supplemented with chemically defined feed and hydrolysate.

Culture medium containing L-fucose - L-fucose (Sigma Cat#F2252) at a concentration of 1-10 mM was added to the base medium on days 0, 3, 6 or 10.

Culture medium containing nicotinamide - Nicotinamide (Sigma Cat#N5535) at a concentration of 0.5-10 mM was added to the base medium on days 0, 3 or 6. Specifically, the feed culture medium contains 0.22 mM of nicotinamide. The detection limit for determining nicotinamide was 20 mg/kg or 0.16 mM nicotinamide.

Antibody purification - Purification was carried out using Protein A, cation and mixed mode chromatography.

Glycan profile analysis - Glycan profile was analyzed by CE-LIF, Beckman coulter / LabChip GXII. Clarified harvest samples were purified and concentrated on MabSelect Sure resin so that their concentration was at least 7mg/mL. 8 μΐ_^ of each protein sample were denatured; glycans were cleaved and labeled using a commercial kit (cat #760523, Perkin Elemer). Samples were run on a Caliper LabChip GXII system using a HT high resolution chip (cat# 760524, Perkin Elemer).

Fc RIIIa binding assay - FcyRIIIa binding assay is based on label free BLI technology with the Octet QK384 system (Fortebio) - an optical analytical technique that measures interference patterns between waves of light. Monitoring the interference pattern in real time provides kinetic data on molecular interactions. In this assay His tagged-FcyRIIIa binds to anti-penta His capture biosensor. Binding kinetics of mAb, at seven concentrations, to bound FcyRIIIa is measured. Resulting signals are converted to affinity parameters by the Octet analysis software. The responses obtained at steady- state for all concentrations were plotted against tested concentrations. Relative binding was calculated relative to the standard sample using the PLA software with a linear regression (parallel line model). Relative binding results, calculated from response curves in the PLA software were found to be a suitable parameter for ranking samples. Therefore, this parameter was used to establish a correlation to percent afucosylated glycans in mAb 18 samples.

Relative efficacy - maximum response ADCC of sample/maximum response ADCC of reference (max response is parameter D on softmax analysis).

ADCC activity assay - ADCC activity was tested using the Promega ADCC Reporter Bioassay. This kit uses engineered Jurkat cells as effector cells. These cell stably express the FcyRIIIa receptor, VI 58 (high affinity) variant and an NFAT response element driving expression of firefly luciferase. Binding of the effector cells to the target through the antibody bridge initiates a cascade of events in the NFAT pathway, resulting in the expression of the firefly luciferase protein. The enzymatic reaction produces luminescence, which is proportional to the luciferase concentration, directly correlating to ADCC activity. ADCC activity is analyzed using a 4- parameter fit where relative potency is calculated from EC50s (parameter C) and relative efficacy, which is mainly influenced by variability in glycan composition, from curve tops (parameter D).

EXAMPLE 1

THE EFFECT OF SUPPLEMENTING THE CULTURE MEDIA WITH L-

FUCOSE ON THE GLYCOSYLATION PATTERN OF ADALIMUMAB

BIOSIMILAR (mAbl8)

Two lots of mAb 18 (adalimumab produced by InSight) presented very high (142 %-173 %) levels of antibody dependent cell mediated cytotoxicity (ADCC) compared to Humira® (data not shown). ADCC is mediated by antibody fucosylation (e.g. a completely afucosylated mab is 50-100 fold more potent than a fucosylated antibody) and these lots of mAbl8 contained higher percent of afucosylated forms (i.e. Man5 and GO) compared to Humira®.

To test the effect of L-fucose on the glycosylation pattern, in general, and the fucosylation pattern, in particular, of mAbl8, mAbl8-expressing CHO cells were cultured in shake flasks (SF) and the culture media was supplemented with different concentrations of L-fucose at different stages of the production process. Without being bound by theory, the rationale of L-fucose addition to the production process was to increase concentration of GDP-fucose in the cell cytosol through the salvage biosynthetic pathway. GDP-fucose is the substrate for fucosyltransferase, an enzyme responsible for glycoprotein fucosylation. As presented in Table 1 below, the addition of 6 mM L-fucose on day 0 or 3 decreased the level of GO (from ~ 4 % to ~ 3 %) and had no effect on other glycoforms. Moreover, addition of higher concentrations of L- fucose did not significantly increase the effect on the level of the GO glycoform.

Table 1. Glycans profile of mAbl8 produced in media supplemented with different concentrations of L-fucose on different days (analyzed by CE-LIF, Beckman Coulter) mAbl8 clarified harvest samples were purified and concentrated on MabSelect Sure resin. The protein samples were de-glycosylated and released glycans labeled by a Prozyme GlykoPrep Kit (Prozyme, cat# GP96NG-AB). A ProteomeLab PA800 Plus (Beckman Coulter) with a neutral capillary (60.2 cm, 50 cm effective length, 50 μιη I.D., Beckman Coulter) was used with constant voltage at 30 kV, reverse polarity and a 0.17 ramp time. The excitation was at 488 nm, emission at 520 nm. The data collection rate was 16 Hz and the dynamic range was set to 100 RFU).

Media A and media B stand for two different mixes of serum-free media. All glycoforms with "F" after name signify the fucosylated form.

L-Fucose addition Glycoforms

Media (mM) (day) Man-5 GO GOF Peak#4 G1F G1'F G2F

Media A 0 9.80% 4.17% 52.32% 2.82% 20.46% 7.32% 3.11%

Media A 1 3 9.57% 3.53% 53.28% 2.67% 20.59% 7.32% 3.04%

Media A 10 3 8.47% 3.13% 54.50% 2.22% 21.00% 7.33% 3.36%

Media B 0 10.67% 3.75% 66.57% 1.37% 12.19% 4.40% 1.05%

Media B 3 0 10.94% 3.40% 66.87% 1.33% 12.01% 4.37% 1.08%

Media B 6 0 10.54% 2.90% 67.67% 1.30% 12.10% 4.45% 1.05%

Media B 10 0 10.28% 2.65% 67.48% 1.28% 12.59% 4.61% 1.12%

Media B 1 3 8.69% 3.60% 62.99% 1.19% 16.02% 5.49% 2.02%

Media B 6 3 10.69% 2.44% 67.78% 1.28% 12.20% 4.54% 1.07%

Media B 10 3 10.33% 2.60% 67.48% 1.23% 12.61% 4.61% 1.15%

Media B 10 6 8.45% 3.66% 61.21% 1.35% 17.22% 5.87% 2.24%

Media B 8 3-10 8.14% 3.42% 62.14% 1.28% 17.04% 5.76% 2.22%

Media B 8 3-10 9.62% 3.03% 68.99% 1.04% 12.04% 4.24% 1.04%

EXAMPLE 2

THE EFFECT OF SUPPLEMENTING THE CULTURE MEDIA WITH NICOTINAMIDE ON THE GLYCOSYLATION PATTERN OF ADALIMUMAB

BIOSIMILAR (mAbl8)

To test the effect of nicotinamide on the glycosylation pattern, in general, and the fucosylation pattern, in particular, of mAbl8, mAbl8-expressing CHO cells were cultured in shake flasks (SF) or 3 liter bioreactor and the culture media was supplemented with different concentrations of nicotinamide at different stages of the production process. As presented in Table 2 below and Figure 1, addition of nicotinamide lead to a decrease in the level of both GO and Man-5 glycoforms in a dose dependent manner, resulting in an overall substantial reduction of afucosylated glycoforms. Moreover, the effect of nicotinamide on mAbl8 glycosylation was correlated with the time point at which it was added to the culture media, i.e. the effect was lower when it was added later during the process. For example, 6 mM of nicotinamide added on day 0 reduced GO by 50 % (from 6 % to 3 %) as compared to a 40 % reduction (to 3.64 %) when the nicotinamide was added on day 3 and only a 22 % reduction (to 4.72 %) when the nicotinamide was added on day 10.

In the next step, samples of mAbl8 produced in media supplemented with the different concentrations of nicotinamide were partially purified and tested for FcyRIIIa binding and ADCC activity. As shown in Figures 2 and 3, addition of nicotinamide to the culture medium reduced both FcyRIIIa binding and ADCC activity of mAbl8 in a dose dependent manner. Furthermore, Figure 3 presents the correlation between nicotinamide concentration, the level of GO, and ADCC activity when nicotinamide was added on days 0 or 3.

Taken together, FcyRIIIa relative response, ADCC efficacy and potency (not shown) showed strong correlation with the level of GO glycoform which decreased with increased nicotinamide concentration in the production medium.

Table 2. Glycans profile of mAbl8 produced in media supplemented with different concentrations of nicotinamide (analyzed by LabChip GXII

Table 3. Glycans profile of mAbl8 produced in 3L bioreactor in media supplemented with different concentrations of nicotinamide (analyzed by LabChip GXII)

Niacinamide Man- day P#0 GO G0F Gl GIF GI F G2 G2F (mM) 5

66.2- 9.7- 2.7- 0.4-

0 0-0.7 7.4-8 6.2 1.6-2 0

71.4 12.7 4.1 0.9

2 0 0.5 6.6 4.8 70.4 1.6 11.7 3.7 0 0.7

6.8- 3.5- 75.3- 1.2- 9.2- 2.6- 0.4-

4 0 0-0.5 0

7.3 3.6 75.5 1.3 9.7 2.7 0.6

5 0 0.5 6.8 3.4 74.9 1.1 9.8 2.9 0 0.6

6 0 0.0 6.6 3.0 78.1 0.9 8.6 2.6 0 0.3 EXAMPLE 3

EXEMPLARY PRODUCTION PROCESS OF mAbl8 IN CULTURE MEDIA

CONTAINING NICOTINAMIDE

mAbl8 expressing cells are thawed into basal culture medium and then propagated in shake flasks for 14-22 days. On day 0 of the production process, 0.3xl0⁶cells/ml are seeded in a bioreactor in basal production medium.

The concentration of nicotinamide in the basal production media is below detection limit (the detection limit is 20 mg/kg or 0.16 mM).

On day 3, 5mM of nicotinamide is added to the basal production media.

From day 3 to day 10 the culture is supplemented by a feed solution that contains a total 0.22 mM of nicotinamide.

The medium containing the secreted antibodies is harvested on day 12-14.

At the end of the process, 3.25 mM of nicotinamide was measured in the clarified harvest.

Thus, theoretically during the culture of mAbl8 nicotinamide is added to the culture in a concentration of 5.22mM.

The range of nicotinamide concentration in the culture during days 3 -end of the process is between 5.38 mM (5.22+the detection limit 0.16) and 3.25 mM.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

WHAT IS CLAIMED IS:

1. A method of altering a property of a recombinant antibody comprising:

(a) analyzing the property of the antibody;

(c) culturing cells which express the recombinant antibody selected in step (b) in a first culture medium comprising a first concentration of nicotinamide, said first concentration being between 0.5 mM - 10 mM nicotinamide, thereby altering the property of the antibody, wherein the property is selected from the group consisting of ADCC activity, glycan profile and Fc-gamma receptor (FcyR) binding.

2. The method of claim 1, further comprising isolating the antibody.

3. The method of claim 2, further comprising testing the amount of fucosylated and/or afucosylated glycoform present in the isolated antibody.

4. The method of claim 1, further comprising culturing cells which express the recombinant antibody in a second culture medium comprising a second concentration of nicotinamide, said second concentration being non-identical to said first concentration of nicotinamide, said second concentration being between 0.5 mM - 10 mM.

5. The method of claim 1, wherein said activity comprises an ADCC activity.

6. The method of claim 4, wherein said afucosylated glycoform is GO or

Man5.

7. A method of generating an antibody comprising:

(b) isolating the antibody; and subsequently (c) testing the amount of fucosylated and/or afucosylated glycoform present in the isolated antibody, thereby generating the antibody.

8. The method of claim 7, wherein said first culture medium comprises between 3-6 mM nicotinamide.

9. The method of claims 7 or 8, wherein said cells are cultured for at least two days in a second culture medium which comprises less than 0.5 mM nicotinamide prior to said culturing.

10. The method of any one of claims 7-8, wherein said culturing is effected for at least 6 days.

11. The method of any one of claims 7-10, wherein said cells comprise CHO cells.

12. The method of any one of claims 7-11, wherein said antibody is a monoclonal Ab (mAb).

13. The method of any one of claims 7-12, wherein said antibody binds tumor necrosis factor (TNF).

14. The method of any one of claims 7-13, wherein the heavy chain of the antibody has an amino acid sequence as set forth in SEQ ID NO: 1 and the light chain of the antibody has the amino acid sequence as set forth in SEQ ID NO: 2.

15. The method of claims 2 or 7, further comprising testing the activity of the antibody following said isolating.

16. The method of claim 7, wherein said afucosylated glycoform is GO or

Man5.

17. The method of any one of claims 7-14, wherein said first culture medium further comprises L-fucose.

18. The method of claim 17, wherein a concentration of L-fucose is between 1-10 mM.

19. The method of claim 18, wherein said concentration of L-fucose is about

6 mM.

20. The method of claim 17, wherein said second culture medium is devoid of L-fucose.

21. A method of generating an antibody comprising culturing cells which express a recombinant antibody in a first culture medium comprising at least 1 mM L- fucose, thereby generating the antibody.

22. The method of claim 21, wherein said first culture medium comprises between 1-10 mM L-fucose.

23. The method of claims 21 or 22, wherein said first culture medium comprises about 6 mM L-fucose.

24. The method of claims 21 or 22, wherein said cells are cultured for at least two days in a second culture medium which is devoid of L-fucose prior to said culturing.

25. The method of any one of claims 21-24, wherein said culturing is effected for at least 6 days.

26. The method of any one of claims 21-25, wherein said cells are CHO cells.

27. The method of any one of claims 21-26, wherein said antibody is a monoclonal Ab (mAb).

28. The method of any one of claims 21-27, wherein said antibody binds tumor necrosis factor (TNF).

29. The method of any one of claims 21-28, wherein the heavy chain of the antibody has an amino acid sequence as set forth in SEQ ID NO: 1 and the light chain of the antibody has the amino acid sequence as set forth in SEQ ID NO: 2.

30. The method of any one of claims 21-29, further comprising isolating the antibody.

31. The method of claim 30, further comprising testing the activity of the antibody following said isolating.

32. The method of claim 30, further comprising testing the amount of GO glycoform present in the antibody.

33. An antibody generated according to the method of any one of the claims 21-30, having lower amounts of GO glycoform as compared with an antibody having been produced in cells which were not cultured in media comprising L-fucose at a concentration greater than 1 mM L-fucose.

34. The antibody of claim 33 having a heavy chain amino acid sequence as set forth in SEQ ID NO: 1 and a light chain amino acid sequence as set forth in SEQ ID NO: 2.

35. A cell culture medium comprising at least 0.5 mM nicotinamide and between 1-10 mM L-fucose.