1. Introduction
Biologic pharmaceuticals are gaining in both market share and clinical utility compared with small molecule therapeutics (Projan et al., 2004). Global biologic drug sales reached $93 billion in 2009 and the sales are expected to grow at least twice as fast as those of small molecules (McCamish and Woollett, 2011). The rapid market growth and the promise of successful rate of developing biologic drugs has drawn the attention of traditional small molecule pharma into the biotechnology business. Today, more than ever, large pharmaceutical companies are venturing into the biotechnology arena with the hope that novel therapeutic proteins will augment the traditional pharmaceutical business enough to fundamentally reshape the market landscape. The acquisition/merging of pipeline and research capacity of biotech companies by big pharma is a great example of this trend. Several companies are even projecting optimistically that, within the decade, therapeutic biologics will comprise a majority of their commercial portfolios (Zhou, 2007). Among highly successful biologic products, monoclonal antibodies (mAbs) are the largest and fastest growing segment. MAbs are established as a key therapeutic modality for a range of diseases most notably rheumatoid diseases and various cancers. Due to the high degree of selectivity of these agents, in particular for cancer targets, they can be designed to selectively target tumor cells and elicit a variety of responses. These agents can kill cells directly by carrying a toxic payload to the target or can orchestrate the destruction of cells by activating immune system components, blocking receptors or sequestering growth factors (Nicolaides et al., 2010).
It is well known that glycosylation can impact the pharmacokinetics, efficacy and tissue targeting of therapeutic proteins (Li and d'Anjou, 2009). N-glycosylation of immunoglobulin G (IgG) at asparagine residue 297 plays a role in antibody stability and is required for immune cell-mediated Fc effector function (Correia, 2010;Kayser et al., 2011;Mimura et al., 2000). Preclinical and clinical studies have demonstrated that modulating the glycosylation of antibody and non-antibody therapeutics can be an effective means to improve the properties of biologic medicines leading to a class of drugs termed "biobetters"(Jefferis, 2009; Walsh, 2010). The strategy of engineering expression hosts to express glycoproteins with "optimized" glycosylation has been applied in both prokaryotic and eukaryotic cells (Beck et al., 2010;Hamilton et al., 2006;Hamilton and Gerngross, 2007;Jacobs and Callewaert, 2009b;Pandhal and Wright, 2010;Tomiya, 2009). Chinese Hamster Ovary (CHO) cells, a widely used host for producing therapeutic antibodies with similar human glycosylation, have been engineered in multiple ways to eliminate the core fucose on secreted mAbs. Recent preclinical and clinical studies have reported superior efficacy utilizing afucosylated mAbs(Herbst et al., 2011;Junttila et al., 2010;von Horsten et al., 2010;Ward et al., 2011;Wong et al., 2010). Non-mammalian expression hosts have also been utilized for producing afucosylated mAbs including insect cells, plants and yeast (Gasdaska et al., 2007;Barbin et al., 2006;Zhang et al., 2011).
Following marketing of the first therapeutic antibody, Muromonab-CD3 in 1986, mAbs used in the clinic have evolved over the years from entirely murine to mouse-human chimeric, and then to humanized and finally fully human antibodies in order to minimize anti-drug related immunogenicity in patients and maintain maximum potency (Li and Zhu, 2010). Several different antibody discovery technologies co-exist today in therapeutic antibody development ranging from isolating antibodies from immunized mice or engineered mice carrying human Ig-repertoire genes to flow-cytometric isolation of human antibodies from non-immune yeast display or panning a large non-immunized phage display libraries (Feldhaus et al., 2003;Vaughan et al., 1996;Weaver-Feldhaus et al., 2004). Recently
Yeast has been widely used for expressing proteins in research and development. There are multiple advantages to using yeast as an expression system for therapeutic glycoprotein production, including ease of genetic manipulation, stable expression, rapid cell growth, high yield of secreted protein, low-cost scalable fermentation processes and no risk of human pathogenic virus contamination. However, glycoproteins expressed in wild type yeast generally cannot be used for therapeutic applications due to fungal type high-mannose glycans which can result in immunogenicity and poor PK
2. Glycosylation in therapeutic proteins is important for its efficacy, PK, tissue targeting
2.1. Glycosylation on Erythropoietin affects its potency
Recombinant human Erythropoietin (rHuEPO) is a 30.4 kDa glycoprotein hormone containing multiple N-linked glycosylation sites and currently is used to treat patients with anemia. The marketed forms of recombinant erythropoietin include Epogen with three native N-glycan structures and Aranesp® (darbepoetin), an epoetin engineered to contain two additional N-glycosylation sites, conferring greater metabolic stability
2.2. Glycosylation on recombinant human glucocerebrosidase is critical for its targeting in enzyme replacement therapy
Gaucher's disease is a lysosomal storage disorder caused by mutations of glucocerebrosidase (GCD), and an enzyme replacement treatment has been developed using recombinant GCD. GCD is glycoprotein and its glycosylation plays an important role in its targeting in therapeutic setting. GCD produced in Chinese Hamster ovary (CHO) cells contains major complex glycan but it has failed to provide clinical benefit in direct infusion due to the preferential uptake of enzyme by hepatocytes rather than Kupffer cells. An
2.3. Afucosylated antibody has enhanced ADCC and can translate into better efficacy
Antibody Dependent Cell-Mediated Cytotoxicity (ADCC) is a mechanism of cell mediated immune defense whereby an effector cell of the immune system actively lyses a target cell that has been bound by specific antibodies. The typical ADCC involves activation of effector cells such as Natural Killer (NK) cells by antibodies. An NK cell expresses Fc receptor IIIa or CD16a (Nimmerjahn and Ravetch, 2007;Nimmerjahn and Ravetch, 2008), and this receptor recognizes and binds to the Fc portion of an antibody which it has already bound to the surface of a pathogen-infected target cell or tumor cell. The NK cell releases cytokines such as IFN-γ, and cytotoxic granules containing perforin and granzymes that enter the target cell and promote cell death by triggering apoptosis. Monoclonal antibody IgG1 Fc has single N-linked site at N-297 and its glycosylation is involved with antibody and Fc γ receptor III binding. Antibodies lacking core fucosylation show a large increase in affinity for Fc γ RIIIa leading to an improved receptor-mediated effector function. The structure study reveals that a unique type of interface consisting of carbohydrate-carbohydrate interactions between glycans of the receptor and the afucosylated Fc. In contrast, in the complex structure with fucosylated Fc, these contacts are weakened or nonexistent (Ferrara et al., 2011). Although afucosylated IgGs exist naturally, a next generation of recombinant therapeutic, glycoenginereed antibodies with enhanced ADCC activity is currently being developed for better efficacy(Mori et al., 2007;Ysebaert et al., 2010;Robak and Robak, 2011).
2.4. Glycans at Fc region impact on its pharmacokinetics
Glycosylation at mAb Fc regions not only plays a role in its effector functions, but it also can impact its pharmacokinetics. The neonatal Fc receptor (FcRn) has a major role in prolonging the exposure of therapeutic mAbs. MAbs internalized by fluid-phase or receptor-mediated endocytosis can be redirected to the cell surface by FcRn mediated recycling and released into plasma or interstitial fluids thus preventing lysosomal degradation(Chowdhury and Wu, 2005; Roopenian and Akilesh, 2007). This recycling dramatically increases systemic exposure to therapeutic mAbs by studies comparing in wild type and FcRn-deficient mice which demonstrated over an order of magnitude increase in IgG half-life as a consequence of the FcRn salvage pathway. Another mechanism for mAb clearance may be through Fc γ receptor (FcγR) binding on some immune effector cells. Interactions with Fc γ receptors can be influenced by the glycosylation pattern. To produce mAbs in lower eukaryotic hosts such as yeast have been reported, however, wild type yeast host expressed antibody secretes full length mAbs with hyper-mannose type glycans. MAb produced in wild type yeast exhibited 2 to 3-fold faster clearance, which might be due to the high mannose content interacting with mannose receptors. On the other hand,
3. Glycoengineered Pichia expresses protein with human glycosylation
3.1. Difference of glycosylation pathway between human and yeast
Yeast N-glycosylation is of the high-mannose type, which differs from human complex glycans. Fungal type of glycans confers a short half-life
3.2. Genetic manipulation of Pichia glycosylation pathway
The process involved eliminating endogenous yeast glycosylation pathways, and we introduced more than 14 heterologous genes into
4. Alternative monoclonal antibody production in glycoengineered Pichia pastoris
4.1. Selection of glycoengineered Pichia expressing mAbs
Conventional mammalian cell lines as expression host secrete glycoprotein usually containing heterogeneous glycans. Mammalian cells, such as Chinese Hamster Ovary (CHO) cells maintain the capability of adding sialic acid at its galatose terminal glycans. However, mAbs expressed in CHO have little or no sialylation at its Fc region due to the steric hindrance of the Fc structure (Nimmerjahn and Ravetch, 2010). On the other hand, glycoengineered
4.2. Difference in glycosylation profile of antibodies from CHO and glycoengineered Pichia
Mammalian cells, e.g. CHO produced mAbs carry N-linked carbohydrate structures with predominantly core-fucosylated asial-biantennary types with varying degrees of galactosylation. Within a given product, there are lot-to-lot variations even though manufacturing processes are tightly controlled and ensure a high degree of product consistency. Besides complex glycoforms, CHO cells expressed monoclonal antibodies still contains some percentage of Man5 type of glycans. While production of consistent and reproducible mAb glycoform profiles still remains a considerable challenge for CHO cells, variations in cell culture processes play a role in the mAb glycosylation profile. Potential variables in the cell culture physicochemical environment including culture pH, cell culture media composition, raw materials lot-to-lot variations, equipment, and process control differences are just a few examples that can potentially alter glycosylation profiles. In a case study, glycoengineered
4.3. Bioanalytical characterization of glycoengineered Pichia produced antibody
A glycoengineered
4.4. Glycoengineered Pichia produced antibody is comparable to CHO derived antibody in vitro assays
Glycoengineered
4.5. Glycoengineered Pichia produced mAb has similar in vivo efficacy and serum half-life
As stated before, fungal type hypermannosylated glycans at the antibody Fc region could have detrimental effects on its pharmacokinetics and would result in fast clearance in the blood stream (Liu et al., 2011). These non-human glycans can trigger the human immune response and causes immunogenicity. To ensure antibody produced from glycoengineered
4.6. Antibody produced by glycoengineered Pichia has better efficacy
Preclinical studies have shown that antibody dependant cell-mediated cytotoxicity (ADCC) is an important part of mechanism of action of therapeutic monoclonal antibodies, especially anti-cancer antibodies, such as Trastumab and Rituximab against tumors (Mori et al., 2007). Some clinical evidence based on genetic analysis of leukocyte receptor (FcγR) polymorphisms of cancer patients treated with anti-CD20 IgG1 Rituximab and anti-HER2 IgG1 Trastuzumab therapies has revealed that ADCC is one of the critical mechanisms responsible for the clinical efficacy of these therapeutic antibodies(Musolino et al., 2008;Kim et al., 2006;Cartron et al., 2002). ADCC enhancement technology is expected to be excised in development of "biobetter" therapeutic antibodies with improved clinical efficacy. A strong correlation with Fc γ receptor affinity and antibody binding to FcγRIIIA in particular has shown to positively correlate with ADCC activity. Trastuzumab produced as Fc engineered or afucosylated mAb showed increased ADCC and improved tumor inhibition in a mouse xenograft model with human immuno-effector cells. A large number of studies with Fc engineered antibodies have firmly established that increased affinity for FcγRIIIA leads to increased NK cell or PBMC-mediated ADCC
5. One stop shop for antibody development with human glycosylation
5.1. Antibody surface display on glycoengineered Pichia
Yeast surface display has been a widely used tool for protein engineering and for antibody engineering in particular (Boder and Wittrup, 1997;Boder and Wittrup, 2000; Wittrup, 2009). However, displaying Fabs or full-length antibodies on the surface of
5.2. Antibody expression platform in glycoengineered Pichia pastoris
Glycoengineered
5.3. Develop a robust and scalable monoclonal antibody production platform using glycoengineered Pichia
Glycoengineered
6. Summary
Glycosylation on therapeutic glycoproteins plays a critical role on its pharmacokinetics, efficacy and tissue targeting. By eliminating pathways responsible for fungal glycosylation and engineering in human glycosylation pathways, glycoengineered
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