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GENERATION OF AN INDUSTRIALLY IDEAL HOST CELL LINE FOR PRODUCING COMPLETELY-DEFUCOSYLATED ANTIBODY WITH ENHANCED ANTIBODY-DEPENDENT CELLULAR CYTOTOXICITY (ADCC)

Mitsuo Satoh, Naoko Yamane-Ohnuki, Katsuhiro Mori, Ripei Niwa, Toyohide Shinkawa, Harue Imai, Reiko Kuni-Kamochi, Ryosuke Nakano, Kazuya Yamano, Yutaka Kanda, Shigeru Iida, Kazuhisa Uchida, Kenya Shitara

Tokyo Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6 Asahi-machi, Machida-shi, Tokyo 194-8533, Japan

Abstract: To generate industrially applicable new host cell lines for antibody production with optimizing antibody-dependent cellular cytotoxicity (ADCC) we focused on the most important carbohydrate structure "fucose residues attached to the innermost GlcNAc residue of N-linked oligosaccharides via a-1,6 linkage" (Shields, 2002; Shinkawa, 2003), and succeeded in disrupting both FUT8 (a-1,6-fucosyltransferase gene) alleles in Chinese hamster ovary (CHO) cell line by sequential homologous recombination. FUT8-/- cell lines have morphology and growth kinetics similar to those of the parent. Antibodies produced by the engineered CHO cell lines strongly bound to human Fcy receptor Ilia (FcyRIIIa) and showed approximately two orders of magnitude higher ADCC than anti-CD20 antibodies (RituxanTM) produced by parental cell lines without changing antigen-binding and complement-dependent cytotoxicity (CDC). Moreover, the engineered cell line remains stable, producing completely-defucosylated antibody with fixed quality and efficacy even in serum-free fed-batch culture. Thus, our approaches provide a new strategy for controlling the glycosylation profile of therapeutic recombinant proteins and could be a considerable advantage for the manufacture of glycoprotein therapeutics, especially antibodies.

Key words: recombinant antibody production; antibody-dependent cellular cyto-toxicity (ADCC); a-1,6-fucosylation; FUT8; siRNA; knockout; fed-batch culture; Chinese hamster ovary (CHO) cells

Introduction

Monoclonal recombinant antibodies IgG, having two N-linked oligosaccharides in the Fc, are commonly used therapeutically. The general structure of IgG N-linked oligosaccharide is complex-type, characterized by a mannosyl-chitobiose core with or without bisecting N-acetylglucosamine (GlcNAc)/L-fucose and other chain variants including the presence or absence of galactose and sialic acid. Although the nature and importance of the oligosaccharides in influencing antibody effector functions was long recongnized (Ripka, 1986; Kumpel, 1995; Jefferis, 1998; Umana, 1999a; Davies, 2001), the most important carbohydrate structure on antibody-dependent cellular cytotoxicity (ADCC) has just recently been found to be fucose residues attached to the innermost GlcNAc residue of N-linked oligosaccharides via a-1,6 linkage (Nanai, 2000; Shields, 2002; Shinkawa, 2003).

ADCC, a lytic attack on antibody-targeted cells, is triggered after binding of lymphocyte receptors (FcyRs) to the antibody constant region (Fc), and is largely dependent on the Fc oligosaccharide structure. Clinical studies have shown that antibody effector functions, especially ADCC, are very important for clinical efficacy (Lewis, 1993; Clynes, 2000). Patients, suffering Non-Hodgkin's lymphoma, having the FcyRIIIa allotype with higher affinity to anti-CD20 IgG1 (Rituxan™) showed the better clinical response than patients with another allotype (Carlton, 2002). Breast cancer patients with complete or partial remission treating with anti-Her2 IgG1 (HerceptinTM) were found to have a higher capability to mediate in vitro ADCC of Herceptin™ (Gennari, 2004). Recently, we demonstrated that ADCC is almost solely controlled by the absence of fucose on IgG1, and that galactose and bisecting GlcNAc contribute little or nothing to ADCC (Hanai, 2000; Shinkawa, 2003). Shields et al. confirmed this observation (Shields, 2002). Defucosylated Rituxan™ shows over 50-fold greater ADCC compared to fucosylated RituxanTM and defucosylated HerceptinTM shows enhanced ADCC with improved FcyRIIIa binding. There is no need to concern the immunogenesity of defucosylated form IgG since it is a normal component of natural human serum IgG (Mizuochi, 1982; Harada, 1987). Thus, IgG1 defucosylation is a powerful and elegant mechanism to improve antibody effector function.

In this review, we describe technologies for recombinant antibody production of biologically-active defucosylated form with enhanced ADCC from several aspects, i.e., useful host cell lines, robustness of production processes, and conversion of established antibody-producing cells. Finally, an industrially-ideal production system for completely-defucosylated therapeutic antibody and its impacts on therapeutic fields are discussed.

Antibody has an unusual structure as glycoprotein

In a general concept, carbohydrates on glycoproteins appear to cover their protein portion just like a cachet. However, the carbohydrates play multiple roles, including not only presentation of binding motifs for lectins and/or protection from proteolytic degradation but also stability of the glycoprotein by directly interacting with polypeptide (Wormald and Dwek, 1999; van Zuylen, 1997). The oligosaccharide of IgG is linked to Asn297 in the Fc region where FcyRIIIa expressed on effector cells such as natural killer cells binds to. The Asn297-linked oligosaccharide contains a mannosyl-chitobiose core (Man3-GlcNAc2), to which fucose, GlcNAc, galactose, mannose, and/or sialic acid are attached (Fig. 1). The crystal structure of IgG Fc region shows that the two oligosaccharides occupy the space between the CH2 domains and extensively contact with the polypeptides (Huber, 1976). Although there is no direct interaction of the carbohydrate with FcyR, complete removal of the Asn297-linked oligosaccharides extinguishes IgG binding to FcyRs, resulting in loss of ADCC (Tao and Morrison, 1989; Radaev, 2001). The unique IgG structure, the location of carbohydrates within protein portion, affects the structure of Fc region and causes the change of antibody effector function ADCC. The antibody defucosylation could contribute to conformational change of the CH2 region to enhance FcyRIIIa binding (Okazaki, 2004).

Defucosylation
Fig. 1. Oligosaccharide structure of IgG1. Complex-type N-linked oligosaccharide (consists of GlcNAc (O), mannose (□), bisecting GlcNAc (•), fucose (★) galactose(n), sialic acid (A)) is attached to the CH2 domains of Fc region IgG1.

Defucosylated antibody production

Recombinant protein expression technology in mammalian cell culture is the principal means of commercial production of therapeutic antibodies; indeed, all approved therapeutic recombinant antibodies are produced using mammalian cells such as Chinese hamster ovary (CHO) and mouse myeloma host cell lines (Table 1). Robust antibody production processes using these host cell lines have been developed. These processes are proven to produce safe and effective antibody molecules with serum half-lives equivalent to those observed for naturally-occurring antibodies. However, it is still remained to be solved to control the oligosaccharide structure of the products to maintain product consistency with desired efficacy because the majority of the recombinant antibody generated by mammalian cells is known to be fucosylated (Kamoda, 2004). A strategy for consistently-regulating recombinant product oligosaccharide structure in mammalian cell culture could be a considerable advantage for the manufacture of therapeutic antibodies. Currently, the methods for generating defucosylated antibody are classified into three categories according to the targeting level of defucosylation (Table 2).

In mammals, almost all antibody fucose residues are attached to the innermost GlcNAc residue of N-linked oligosaccharides via an a-1,6 linkage (Miyoshi, 1999). a-1,6-fucosyltransferase catalyzes the transfer of fucose from GDP-fucose to the GlcNAc residue in an a-1,6 linkage in the medial Golgi cisternae. FUT8 encodes an a-1,6-fucosyltransferase gene, and no isoforms have yet been cloned (Uozumi N, 1996). To control the fucosylation of recombinant antibody produced by mammalian cells, FUT8 activity has to be modified somehow. Historically, we had to focus on unique cell line, such as rat hybridoma YB2/0, with reduced intrinsic fucosylation activity (Hanai, 2000). YB2/0 cells, which produce relatively-highly defucosylated (30-50%)

recombinant antibodies, express a more than 10-fold lower level of FUT8 transcripts than CHO cells, and overexpression of FUT8 in YB2/0 cells rescues its a-1,6 fucosylation activity rendering it equivalent to that of CHO cells (~10% defucosylated) (Shinkawa, 2003). In the 1st generation there were limitations of defucosylated level of the products and host cells available. Next, the methods for converting host cell lines or normal antibody-producing cells to more desirable cell lines that have ability to produce 60-90% defucosylated antibody were developed using Lens culinaris agglutinin (LCA)-resistant clones or small interfering RNA (siRNA) against FUT8 (Kanda, 2002; Shields, 2002; Mori, 2004). The FUT8 siRNA-intorduced cells that show resistancy to LCA, which recongnizes the a-1,6 fucosylated trimannose-core structure of N-linked oligosaccharides, can produce the high-ADCC antibody with the oligosaccharide structure equivqlent to the parental one except for the fucose content. Defucosylation level of the product by the siRNA-intorduced cells reaches to approximately 60% and stably lasts at the end of serm-free fed-batch culture, which is very improtant from an industrial application standpoint because manufacture processes must guarantee fixed product properties. Manufacture of a recombinant antibody with structure-desired and consistent carbohydorates is necessary for keeping and controlling therapeutical activity i.e. ADCC of the ingredients.

As an alternative method, overexpression of p-1,4-acetylglucosaminyltransferase III (GnTIII) is reported to indirectly lead to defucosylation of produced antibody with increased bisecting GlcNAc of the oligosaccharide by Dr. Umana (GlycArt Biotechnology AG) in this symposium. The mechanism for the defucosylation induced by GnTIII-overexpression, however, is still remained to be solved because there are some exceptions in which GnTIII hyper-expressing transformed cells mainly produce fucosylated products (Ohno, 1992; Umana, 1999a,b; Davies, 2001; Shinkawa, 2003) and the increase in the content of bisecting GlcNAc is not accompanied by the increase in defucosylation (Hard, 1992; Chen, 1998; Morelle, 2000).

Table 1. Recombinant therapeutic antibodies on the market USA

Product

Comiunv

Type

Antigen

Indication

Host

Approval

ReoPro

Centocor/Lilly

Chimera

gpllb/llla Thrombosis

SP2/0

1994

Rituxan

ll>K('/Ccncntcch/K(>chc

Chimera

CD20

NHL

CHO

1997

Zenepax

Roche

Humanized

IU»

Transplantation

NSO

1997

Rcmkadc Centocor/J&J

Chimera

TNFa

RA, Cron's

SP2/0

1998

Synagis

Med Immune/Abotl

Humanized

RSV

RSV Infection

NSO

1998

Sim ii led

Novartis

Humanized

II.-2K

Transplantation

SP2/0

1998

Hereeptin

(rcncn tech/Roche

Humanized

Her2

Brusl Cancer

CHO

1998

Mylotarg

Celllech/AHP

Drug~conjugale CD33

A ML

NSO

2000

Cnmpath

ILEX/Schering

Humanized

CDSI

B-CLL

CHO

2001

Zcvalin

IDFX/Schertng

WY -conjugale

CD20

NHL

CHO

2002

Il u m ira

A bbolt/CAT

Fully buman

TNFa

RA

CHO

2002

Beuitr

Coriia/SKB

1J,l-conjugate

CD20

NHL

CHO

2003

X u lair

Giftentech/NovaHU/Tanox Humanized

IgE

Allergic asthma

CHO

2003

Generation of an Industrially Ideal Host Cell Line Table 2. Defucosylated antibody production methods

1" Generation - Production of 30-50% fucose negative antibody

• Use cell lines with reduced intrinsic fucosylation activity

2"a Generation - Production of 60-90% fucose negative antibody

• Generation of lectin-resistant cell lines, FUT8 RNAi-introduced cell lines from host cells or antibody producing cells

3rd Generation - Production of 100% fucose negative antibody

• Host cells that are incapable adding fucose, because of FUT8 knock out.

An ideal host cell line for completely-defucosylated antibody

Ideally completely-defucosylated antibody should be supplied to patients as therapeutics simply because biological activity of antibody is largely dependent on the fucosylation level and completely-defucosylated antibody shows more potent efficacy both in vitro and in vivo (Niwa, 2004a,b). Industry cannot keep from taking into consideration that a stable supply of therapeutic antibody with uniformed and specified biological activity according to regulation. Production system in which the fucosylation ratios of the products vary in processes depending on the producing clones and/or the culture condition probably causes the fatal problem in terms of industrial application. For the purpose of stable supply of therapeutic antibody with defucosylation, complete and irreversible inactivation of FUT8 function in producing cells is essential. Thus gene targeting, an extremely rare event in somatic cells (Hanson and Sedivy, 1995), is necessary in the cells employed for antibody production. We succeed in the targeted disruption of both FUT8 alleles in host cells CHO/DG44 by homologous recombination to generate new cell lines for production of completely-defucosylated antibody (Yamane-Ohnuki, 2004). FUT8-/- cell lines have morphology and growth kinetics similar to those of the parent. Antibodies produced by the engineered CHO cell lines strongly bound to human FcyRIIIa and showed approximately two orders of magnitude higher ADCC compared to parental antibody RituxanTM without change in antigen-binding and complement-dependent cytotoxicity (CDC) activities. Moreover, the engineered cell line remains stable, producing completely-defucosylated antibody with fixed quality and efficacy even in serum-free fed-batch culture. FUT8cell lines have the great advantage of providing uniformity to biopharmaceutical N-linked glycosylation. FUT8-/- cell lines are expected to achieve both improved efficacy and reduction of dose and cost of therapeutic antibody. FUT8-/- CHO/DG44 cell lines, the ideal host cells to stably produce high-ADCC therapeutic antibodies, are now available.

References

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