Glycoengineering design options for IgG1 in CHO cells using precise gene editing

Glycoengineering design options for IgG1 in CHO cells using precise gene editing Abstract Precise gene editing technologies are providing new opportunities to stably engineer host cells for recombinant production of therapeutic glycoproteins with different glycan structures. The glycosylation of recombinant therapeutics has long been a focus for both quality and consistency of products and for optimizing and improving pharmacokinetic properties as well as bioactivity. Structures of glycans on therapeutic glycoproteins are important for circulation, biodistribution and bioactivity. In particular, the latter has been demonstrated for therapeutic IgG1 antibodies where the core α1,6Fucose on the conserved N-glycan at Asn297 have remarkable dampening effects on antibody effector functions. We previously explored precise gene engineering and design options for N-glycosylation in CHO cells, and here we focus on engineering options possible for N-glycans on human IgG1. We demonstrate stable precise gene engineering of rather homogenous biantennary N-glycans with and without galactose (G0F, G2F) as well as the α2,6-linked monosialylated (G2FS1) glycoform. We were unable to introduce substantial disialylated glycoforms. Instead we engineered a novel monoantennary homogeneous N-glycan design with complete α2,6-linked sialic acid capping. All N-glycoforms may be engineered with and without core α1,6Fucose. The stably engineered design options enable production of human IgG antibodies with an array of distinct glycoforms for testing and selection of optimal design for different therapeutic applications. Chinese hamster ovary, galactosyltransferase, genetic engineering, mAbs, sialyltransferase Introduction Monoclonal antibodies (mAbs) constitute a growing class of recombinant glycoprotein therapeutics and currently represent about a quarter of the approved biologics in the market, and mAbs are currently in use for a variety of infectious, inflammatory, autoimmune and cancer diseases (Walsh 2014). Most therapeutic mAbs are human or humanized IgG1 glycoproteins with a conserved N-glycan at Asn297 in its Fc-region., and the structure of this N-glycan has important roles in modulating interactions with Fcγ receptors on immune cells and complement (Jefferis 2016). Processing of the Asn297 N-glycan beyond the point of first complex-type N-acetylglucosamine residues is generally incomplete. This has been shown to be due to glycan–peptide backbone interactions and steric hindrance for subsequent enzymes to efficiently process the N-glycan, and the Asn297 glycosite in IgG1 represents a classic example of site-specific N-glycostructures driven by local constraints in accessibility (Barb and Prestegard 2011). Thus, the common N-glycan structure found on recombinant expressed IgG1 is based on a biantennary N-glycan scaffold with a core α6Fucose and with low galactose and sialic acid contents (Figure 1A). The importance of the structure of the Asn297 N-glycan for effector functions of therapeutic IgG was originally demonstrated using Lec13 mutant CHO cells or rat hybridoma YB2/0 cells for production of IgG1 without the core α6Fucose, and such IgG mAbs exhibited 50-fold increased binding to FcyRIIIa and higher antibody-dependent-cell-cytotoxicity (ADCC) (Shields et al. 2002; Shinkawa et al. 2003). This discovery paved the way for several cancer therapeutic IgG1 antibodies with improved efficacy in the clinic, including Mogamulizumab (Poteligeo) and Obinutuzumab (Gazyva), which are produced in Chinese hamster ovary (CHO) cells genetically engineered to produce N-glycans with no or low fucose by knockout of the core α6fucosyltransferase Fut8 or overexpression of the bisecting β4GlcNAc-transferase MGAT3 (Umaña et al. 1999; Yamane-Ohnuki et al. 2004). Studies have further demonstrated that also galactosylation and sialylation of the Asn297 glycan are important for receptor binding and effector functions (Boyd et al. 1995; Ohmi et al. 2016; Dekkers et al. 2017), indicating that further engineering of the N-glycan structure of therapeutic IgG1 may have great biomedical potential. Fig. 1. View largeDownload slide IgG1 biantennary N-glycan engineering design options. (A) UPLC profiling analysis of N-glycans released from IgG produced in CHOWT shows a heterogeneous mixture of predominant G0F, G1F, and minor amounts of G2F. (B) KI of B4GALT1 (CHO+B4GALT1) results in almost homogeneous G2F. (C) KI of B4GALT1 and ST3GAL4 (CHO+B4GALT1/+ST3GAL4) generates heterogeneous G2F, G2FS1 and G2FS2 glycoforms. (D) KI of B4GALT1 and ST6GAL1 KI (CHO+B4GALT1/+ST6GAL1) gives mainly G2FS1 and minor G2F glycoforms. (E) B4GALT1 and ST6GAL1 KI and KO of St3gal4 and St3gal6 (CHO−St3gal4/St3gal6/+B4GALT1/+ST6GAL1) shows mainly G2FS1 and minor G2F and G2FS2 glycoforms. (F) KO of Fut8 in CHO+B4GALT1/+ST6GAL1 results in complete removal of core α1,6Fucose. Designations for monosaccharides according to the Consortium for Functional Glycomics are indicated. Designations for N-glycan structures Gal (G0–G1–G2), α6Fuc (F), and sialic acid (S1–S2). Fig. 1. View largeDownload slide IgG1 biantennary N-glycan engineering design options. (A) UPLC profiling analysis of N-glycans released from IgG produced in CHOWT shows a heterogeneous mixture of predominant G0F, G1F, and minor amounts of G2F. (B) KI of B4GALT1 (CHO+B4GALT1) results in almost homogeneous G2F. (C) KI of B4GALT1 and ST3GAL4 (CHO+B4GALT1/+ST3GAL4) generates heterogeneous G2F, G2FS1 and G2FS2 glycoforms. (D) KI of B4GALT1 and ST6GAL1 KI (CHO+B4GALT1/+ST6GAL1) gives mainly G2FS1 and minor G2F glycoforms. (E) B4GALT1 and ST6GAL1 KI and KO of St3gal4 and St3gal6 (CHO−St3gal4/St3gal6/+B4GALT1/+ST6GAL1) shows mainly G2FS1 and minor G2F and G2FS2 glycoforms. (F) KO of Fut8 in CHO+B4GALT1/+ST6GAL1 results in complete removal of core α1,6Fucose. Designations for monosaccharides according to the Consortium for Functional Glycomics are indicated. Designations for N-glycan structures Gal (G0–G1–G2), α6Fuc (F), and sialic acid (S1–S2). Genetic engineering of the glycosylation capacity of mammalian cells was for long hampered by the need for successive homologous recombination cycles to knockout unwanted endogenous glycosylation features (Yamane-Ohnuki et al. 2004). With the emergence of the precise gene editing technologies it is now possible to perform combinatorial and comprehensive KO/KI glycoengineering in mammalian cells, and develop host cell lines with a high degree of stable custom designed glycosylation capacity (Yang, Wang et al. 2015). Considerable efforts in the last decades have been devoted to engineering of the N-glycosylation capacities of mammalian, insect, yeast and plant production cells (Hamilton et al. 2006; Durocher and Butler 2009; Clausen et al. 2015), and today it is possible to produce recombinant glycoproteins in all systems with more or less heterogeneous N-glycans compatible with therapeutic use in humans (Hollister et al. 2002; Castilho et al. 2010, Laukens et al. 2015). Engineering the glycosylation capacity of cells for therapeutic IgG has, however, proved more challenging. Overexpression of galactosyltransferases and sialyltransferases in different host cells have demonstrated some increase in the galactosylation and sialylation efficiencies of the Asn297 N-glycan, but the achieved glycosylation effects have been heterogeneous and with limited degree of sialylation (Castilho et al. 2010; Dekkers et al. 2016; Lalonde and Durocher 2017). The mammalian CHO cell is by far the most used production platform for recombinant therapeutics (Walsh 2014), and the genome and endogenous N-glycosylation capacity of CHO cells are well characterized (North et al. 2010; Xu et al. 2011). It has been argued that use of nonmammalian cells with less complex glycosylation may offer better opportunities to rebuild more homogenous human N-glycosylation (Hamilton et al. 2006; Chen 2016, Kallolimath et al. 2016). However, the complex engineering required with de novo expression of entire glycosylation pathways may suffer in stability and consistency in yeast and plant systems (Martínez de Alba et al. 2013; Laukens et al. 2015). The novel opportunities with facile combinatorial precise KO/KI glycoengineering in mammalian cells clearly cement CHO as the preferred host cell line (Yang, Wang et al. 2015). Here, we systematically explored options for stable precise genetic engineering of the N-glycosylation of IgG1 in CHO cells, and we developed a design matrix that enable production of IgG with distinct N-glycan structures including novel and homogeneous monoantennary structure designs. Results The stable genetic engineering strategy We first established CHO clones stably expressing high levels (approximately 300 mg/L in batch culture) of the anti-rabies human IgG1 SO57 (Sealover et al. 2013; Yang, Wang et al. 2015). Glycoprofiling of IgG purified from these cells showed the typical N-glycan structures found on recombinant IgG expressed in CHO cells with heterogeneous G0F and minor amounts G1F and G2F and essentially undetectable nonfucosylated structures (Figure 1A). We next used these IgG expressing CHOWT clones in a combinatorial engineering strategy involving KI of human glycosyltransferase genes to improve galactosylation and alpha2,3sialylation, as well as to introduce the more predominant human α2,6sialylation. We also used KO to disrupt endogenous glycosyltransferase genes in order to reduce heterogeneity and eliminate core fucosylation and biantennary glycans. All KO events were verified by gene sequencing (Supplementary data, Table S1) and unaltered expression of IgG was verified by ELISA. To circumvent stability problems with random plasmid integration, we first used a modified ObLiGaRe strategy (Maresca et al. 2013; Yang, Wang et al. 2015), for targeted KI of codon optimized full coding constructs of human genes B4GALT1, ST3GAL4 and ST6GAL1 into a Safe-Harbor locus, without use of antibiotic selection. The Safe-Harbor locus was recently identified and demonstrated to enable stable transcription during cell culture processes without affecting expression of adjacent genes (Bahr et al. 2016). We designed the donor plasmids for targeted KI with two flanking ZFN binding sites to ensure clean targeting without introducing any backbone sequence of donor plasmid. Furthermore, we included two tDNA insulator elements flanking the transcription unit to minimize epigenetic silencing (Gaszner and Felsenfeld 2006). In order to stack multiple exogenous genes by KI, we applied an alternative strategy using PCR amplicon of the constructs described above together with CRISPR-Cas9 for targeted KI of a second gene into the Safe-Harbor gRNA recognition locus, localized adjacent to the ZFN binding site. KI clones were initially screened by immunocytology with antibodies and lectins (Supplementary data, Figure S1), and correct targeted integration into the Safe-Harbor site was confirmed by junction PCR (Supplementary data, Figure S2). Interestingly, the first screening by immunocytology showed variable and heterogeneous Golgi-like staining in the initial pool of cells, and clones with homogenous and strong staining were selected and found to maintain stable expression of the KI glycosyltransferase genes. Engineering biantennary N-glycoforms on human IgG1 We previously showed that KO of the endogenous B4galt1 galactosyltransferase gene in CHO cells resulted in production of IgG with homogeneous G0F N-glycans (Yang, Wang et al. 2015), and here we found that monoallelic targeted KI of human B4GALT1 (CHO+B4GALT1) resulted in significantly improved galactosylation of IgG and essentially a homogeneous G2F glycoform with very limited sialylation (Figure 1B). To introduce sialylation, we tested KI of human ST3GAL4 for α2,3sialylation and ST6GAL1 for α2,6sialylation, as these have been shown to be the most efficient sialyltransferase isoenzymes acting on N-glycans in CHO (Yang, Wang et al. 2015). Targeted KI of ST3GAL4 into CHO+B4GALT1 to generate CHO+B4GALT1/+ST3GAL4 did improve α2,3sialylation but only partially (Figure 1C). In contrast, monoallelic KI of ST6GAL1 into CHO+B4GALT1 to generate a CHO+B4GALT1/+ST6GAL1 resulted in conversion of the G2F glycoform to a rather homogenous G2FS1 glycoform with minor amounts of G2F and detectable G2FS2 (Figure 1D). To explore potential interference from endogenous α2,3sialyltransferases and ensure that sialylation was homogeneously α2,6-linked, we further stacked KO of the two predominant endogenous sialyltransferase genes, St3gal4 and St3gal6, responsible for sialylation of N-glycans in CHO (Yang, Wang et al. 2015). This did not appear to affect the degree of sialylation in the CHO+B4GALT1/+ST6GAL1 cell line (Figure 1E). The core α6Fucose on N-glycans is controlled by the Fut8 gene, and KO of this gene results in complete loss of α6Fuc (Miyoshi et al. 1999; Yang, Wang et al. 2015). We confirmed this in the CHO+B4GALT1/+ST6GAL1 line by stacked KO of Fut8 (Figure 1F). The KO of Fut8 appeared to lower the efficiency of sialylation slightly, but it is unclear if this is related to the clonal ancestry or it is a general feature. Clearly further studies are needed, but we predict that any of the presented N-glycan designs are largely interchangeable with and without the core Fucose. Engineering monoantennary N-glycoforms on human IgG1 Inspired by our previous studies engineering monoantennary N-glycoforms (Yang, Wang et al. 2015), as well as the GlycoDelete concept (Meuris et al. 2014), we explored monoantennary design options for the conserved N-glycan on IgG by KO of the Mgat2 gene directing biantennary formation (Figure 2A). KO of Mgat2 in CHOWT produced a heterogeneous mixture of monoantennary N-glycans with and without galactosylation and sialylation (G0F, G1F, G1FS1) (Figure 2B). KO of Mgat2 in the B4GALT1 monoallelic KI clone CHO+B4GALT1 to generate CHO−Mgat2/+B4GALT1 resulted in monoantennary N-glycans with complete galactosylation, and interestingly the endogenous α2,3sialylation remained incomplete to generate a mix of monoantennary G1F and G1FS1 (Figure 2C). We further stacked ST3GAL4 by targeted KI into CHO−Mgat2/+B4GALT1 to generate CHO−Mgat2/+B4GALT1/+ST3GAL4, which presented a marked increase in α2,3sialylation, although some nonsialylated G1F remained (Figure 2D). Removal of the endogenous sialylation by KO of St3gal4 and St3gal6 in CHO−Mgat2/+B4GALT1 (CHO−Mgat2/−St3gal4/−St3gal6/+B4GALT1), resulted in homogenous monoantennary N-glycans devoid of sialylation but with complete galactosylation (G1F) (Figure 2E). To obtain homogenous α2,6sialylation, we did targeted KO of St3gal4 and St3gal6 to eliminate α2,3sialylation capacity along with Mgat2 KO into the double KI clone CHO+B4GALT1/+ST6GAL1 (CHO−Mgat2/−St3gal4/−St3gal6/+B4GALT1/+ST6GAL1), which resulted in efficient α2,6sialylation capacity and a homogeneous G1FS1 glycoform (Figure 2F). Fig. 2. View largeDownload slide IgG1 monoantennary N-glycan engineering design options. (A) UPLC profiling analysis of N-glycans released from IgG produced in CHOWT. (B) KO of Mgat2 in CHOWT generates heterogeneous monoantennary G0F, G1F and G1FS1 (CHO−Mgat2). (C) Additional KI of B4GALT1 (CHO−Mgat2/+B4GALT1) results in G1F and G1FS1. (D) Further KI of ST3GAL4 (CHO−Mgat2/+B4GALT1/+ST3GAL4) shows an increase in G1FS1 with 2,3-linked sialic acid but remaining G1F. (E) KO of St3gal4 and St3gal6 in CHO−Mgat2/+B4GALT1 results in homogeneous G1F and (F) additional KI of ST6GAL1 (CHO−Mgat2/−St3gal4/−St3gal6/+B4GALT1/+ST6GAL1) generates homogeneous G1FS1 with 2,6-linked sialic acid. (G) KO of Man2A1 and Man2A2 in CHOWT introduces an alternative monoantennary design with a tri-mannose core on the α6-arm. All glycoforms are predicted to convert to nonfucosylated with KO of Fut8. Fig. 2. View largeDownload slide IgG1 monoantennary N-glycan engineering design options. (A) UPLC profiling analysis of N-glycans released from IgG produced in CHOWT. (B) KO of Mgat2 in CHOWT generates heterogeneous monoantennary G0F, G1F and G1FS1 (CHO−Mgat2). (C) Additional KI of B4GALT1 (CHO−Mgat2/+B4GALT1) results in G1F and G1FS1. (D) Further KI of ST3GAL4 (CHO−Mgat2/+B4GALT1/+ST3GAL4) shows an increase in G1FS1 with 2,3-linked sialic acid but remaining G1F. (E) KO of St3gal4 and St3gal6 in CHO−Mgat2/+B4GALT1 results in homogeneous G1F and (F) additional KI of ST6GAL1 (CHO−Mgat2/−St3gal4/−St3gal6/+B4GALT1/+ST6GAL1) generates homogeneous G1FS1 with 2,6-linked sialic acid. (G) KO of Man2A1 and Man2A2 in CHOWT introduces an alternative monoantennary design with a tri-mannose core on the α6-arm. All glycoforms are predicted to convert to nonfucosylated with KO of Fut8. An alternative approach to generate a monoantennary N-glycan structure was achieved by KO of the mannosidase genes, Man2A1 and Man2A2, in CHOWT (Figure 2G). This resulted in a rather heterogeneous monoantennary structure with an extended mannose structure on the α6-arm and a partially galactosylated and sialylated α3-arm. We predict that further combined KI of B4GALT1 and ST6GAL1 and KO of St3gal4 and St3gal6 would result in a homogeneous monoantennary structure as demonstrated for the engineering sequence based on the Mgat2 KO, but with a more elaborate α6-arm. Targeting ER/Golgi proteins known to effect transport and residency of glycosyltransferases Studies have demonstrated that the stability of Golgi glycosyltransferases, including β4Gal-T1 and ST6Gal-I, may be regulated by resident proteases such as signal peptide peptidase-like 3 (SPPL3) (Voss et al. 2014; Kuhn et al. 2015). We therefore investigated if targeted KO of this protease would improve glycosylation efficiencies in CHO cells. KO of the Sppl3 gene in CHOWT cells appeared to increase galactosylation of IgG1 slightly, however, KO of Sppl3 in the CHO+B4GALT1 and CHO+B4GALT1/+ST6GAL1 KI clones did not substantially affect the glycosylation profile or improve α2,6 sialylation. Similarly, β-site APP cleaving enzyme 1 (BACE-1) has previously been shown to cleave ST6Gal-I (Kitazume et al. 2003), but KO of the Bace1 gene did not appear to affect the sialylation efficiency in the CHO+B4GALT1/+ST6GAL1 clone (not shown), and we therefore did not pursue these engineering strategies further. Evaluation of clonal stability In agreement with our previous study, we found that CHO cells have great plasticity for targeted KO/KI glycoengineering (Yang, Wang et al. 2015), as we did not observe gross effects on viability, growth, and recombinant production of the IgG for any of the genetic designs introduced (Figure 3A). To evaluate this in more detail, we chose the CHO+B4GALT1/+ST6GAL1 double KI cell line and cultured it for 25 passages, which represents at least 50 generations and is beyond the needs for full scale up of antibody production cell lines. The IgG N-glycosylation was identical at passages 1 and 25 (Figure 3B). Fig. 3. View largeDownload slide Stability of glycodesigns. (A) Growth and viability analysis over 8 days for CHOWT and CHO+B4GALT1 clones. The CHO clones were seeded at 0.25 million cells/mL in 50 mL shaking tubes with 30 mL media volume. Mean of three independent cultures. (B) Glycoprofiling by CE-LIF of a CHO+B4GALT1/+ST6GAL1 clone before and after growth over 25 passages in 50 mL shake tubes. Fig. 3. View largeDownload slide Stability of glycodesigns. (A) Growth and viability analysis over 8 days for CHOWT and CHO+B4GALT1 clones. The CHO clones were seeded at 0.25 million cells/mL in 50 mL shaking tubes with 30 mL media volume. Mean of three independent cultures. (B) Glycoprofiling by CE-LIF of a CHO+B4GALT1/+ST6GAL1 clone before and after growth over 25 passages in 50 mL shake tubes. Discussion Here, we continued our glycoengineering efforts using precise gene editing to establish stable CHO cell lines with well-defined N-glycosylation capacities designed specifically for production of therapeutic IgGs. The design matrix presented for stable glycoengineering of CHO cells enables production of IgG antibodies with traditional biantennary G0, G2, G2S1 with and without core fucose. We were unable to introduce the homogeneous disialylated G2FS2 glycoform, and this glycoform has thus far only been achieved by extensive in vitro enzymatic glycosylation (Huang et al. 2012; Washburn et al. 2015). The G2FS2 glycoform has also been produced chemoenzymatically using a mutagenized endoglucosidase (EndoS) with transglycosylation activity to introduce a synthetic biantennary glycans (Huang et al. 2012). A number of studies have shown that the biological activity of IgG1 is influenced by the structure of the Asn297 N-glycan in the Fc region (Wuhrer et al. 2015; Jefferis 2016; Lu et al. 2016). Other N-glycan features influence the flexibility of the Fc domain and affect the interactions with immune receptors. Thus, increased galactosylation improves C1q binding and complement dependent cytotoxicity (CDC) (Dekkers et al. 2017; Peschke et al. 2017), while α2,6sialylation abrogates this effect (Quast et al. 2015). Increased galactosylation has been shown to improve IgG binding to FcγRIIIa and increase ADCC (Thomann et al. 2015), while effects of α2,6sialylation on FcγRIIIa binding is more controversial (Thomann et al. 2015; Lin, Tsai et al. 2015; Dekkers et al. 2017). Increased 2,6-sialylation of intravenous immunoglobulin and IgGs has been shown to induce an anti-inflammatory activity (Kaneko et al. 2006; Anthony et al. 2008; Oefner et al. 2012, Ohmi et al. 2016; Pagan et al. 2018). Although our CHO design matrix allows stable and homogeneous productions of IgG1 to further investigate glycan dependent biological activities, circulating IgG may undergo extracellular sialylation by secreted ST6Gal1 (Jones et al. 2016) and is a possible additional level of regulation of the immunomodulatory functions of IgG. Various strategies have been pursued to produce IgG with different glycoforms. Culture conditions may improve glycosylation efficiencies and especially sialylation slightly, but heterogeneity always remains (Jefferis 2016). The first genetic glycoengineering in CHO cells introduced α2,6sialylation capacity by stable plasmid transfection with ST6GAL1 (Lee et al. 1989), and since then a large number of studies have used overexpression of α2,3- or α2,6-sialyltransferases with or without β4-galactosyltransferases in CHO (Weikert et al. 1999; El Maï et al. 2013; Lin, Mascarenhas et al. 2015; Raymond et al. 2015). Heterogeneity and incomplete galactosylation and sialylation were consistent issues, although one study suggests that overexpression of B4galt1 and St6gal1 in a murine hybridoma cell line may lead to the fully sialylated G2FS2 N-glycan structure on mouse IgG1 (Ohmi et al. 2016). In agreement with these studies, we were able to induce efficient galactosylation (G2F) and sialylation (G2FS1), although significant levels of G2FS2 were not reached, stressing the difficulties with glycosylation of the Asn297 N-glycan (Figure 1). Stable genetic engineering of the glycosylation capacities of mammalian cells have only recently been facilitated by precise gene editing technologies (ZFNs, TALENs and CRISPR/Cas9) (Steentoft et al. 2014). Eliminating unwanted glycosylation capacities is fairly straightforward using KO of individual glycogenes, and stacking multiple targeting events in CHO cells is also now possible (Yang, Wang et al. 2015). Moreover, targeted KI into Safe-Harbor sites enables selection of stable glycosyltransferase KI clones with homogeneous expression levels (Figure 1). Once selected the enzyme expression and glycan profile were stable and consistent beyond the cell generations generally required for industrial scaled fed-batch production (Figure 3). The precise gene engineering enabled us to establish cell lines producing a level of homogeneity in glycosylation of IgG previously unreachable (Figure 1). An elegant example of an entirely novel glycoengineering strategy for IgG1 is presented with GlycoDelete (Meuris et al. 2014), where a Golgi-targeted fungal endoglucosidase (endoT) is expressed in human HEK293 cells to generate simple N-glycans with a heterogeneous mix of mono-, di- or trisaccharide structures (Neu5Ac–Gal–GlcNAc-Asn) resulting in a relative homogeneous Fc-region on an anti-CD20 mAb. Interestingly, the GlycoDelete antibody showed less initial clearance from serum in mice, and a lower binding affinity to human Fcγ receptors. The long circulatory half-life of IgG1 of 3 weeks is largely governed by cellular recycling by the neonatal Fc-receptor (FcRn) that binds independent of the N-glycan (Tao and Morrison 1989; Burmeister et al. 1994). This imply that glycosylation, and in particular sialylation, may not be relevant for circulation in contrast to most other therapeutic glycoproteins (Fukuda et al. 1989). However, truncating the N-glycan by GlycoDelete appears to improve initial circulation somewhat (Meuris et al. 2014). Here, we pursued a different strategy to generate simpler and homogeneous N-glycans on IgG with our monoantennary design (Figure 2), and future studies are needed to explore the biological consequences of these designs. In summary, we presented specific engineering options for the unique N-glycan at Asn297 on human IgG1. While we were unable to engineer the biantennary G2FS2 N-glycoform, we obtained CHO cells with rather homogeneous G2F and G2FS1 glycoforms, as well as novel homogeneous monoantennary G1F and G1FS1 glycoforms. Availability of the design matrix and engineered cells for consistent production of different human IgG1 glycoforms will now enable wider studies and productions of optimal glycoforms for specific therapeutic applications. Materials and methods Recombinant expression of IgG in CHO cells The anti-rabies human IgG1 SO57 (Sealover et al. 2013) was used to establish stable expressing CHO clones. Briefly, we constructed and transfected a pEE-IgG vector (Lonza Biologics) into CHOZN GS−/− cells (Sigma-Aldrich) before glutamine selection was used to select stable IgG expressing clones. All media, supplements and other reagents were obtained from Sigma-Aldrich unless otherwise specified. CHO cells were maintained as suspension cultures in EX-CELL CHO CD Fusion serum-free media, supplemented with 4 mM l-glutamine either in 50 mL TPP TubeSpin® Bioreactors at 200 rpm or 100 mL in Corning 500 mL culture bottle at 130 rpm (Infors), both at 36.5°C and 5% CO2 in air. For IgG production, clones were seeded at 0.5 × 106 cells/mL without l-glutamine and cultured for 3 days for IgG production, and IgG was purified by HiTrapTM Protein G HP (GE Healthcare, US) pre-equilibrated in PBS and eluted with 0.1 M glycine (pH 2.7). Purity of proteins was evaluated by Coomassie staining of SDS-PAGE gels, and proteins were quantified by BCA Protein Assay Kit (Thermo Scientific) or direct ELISA. For stability studies cells were grown in 50 mL tubes with 30 mL media volume, and split (1:8 ratio) two times every week with seeding at 0.25 million cells/mL. Gene targeting in CHO cells Gene targeting by CRISPR/Cas9 and ZFN were performed in CHO cells expressing IgG and 25–100 clones were screened for each targeting event. Cells were seeded at 0.5 × 106 cells/mL 2 days prior to transfection. KO was performed with 1 μg endotoxin-free plasmid DNA of each Cas9-GFP and gRNA in px458 vector (Addgene). KI was performed with 2 μg of each ZFN tagged with GFP/Crimson as previously described (Duda et al. 2014), and 5 μg donor plasmid with full coding codon-optimized human glycosyltransferase genes (B4GALT1, ST3GAL4 or ST6GAL1). To KI a second gene into the Safe-Harbor locus we used 1 μg of a PCR product encoding the codon optimized human enzymes with 1 μg Cas9-GFP and gRNA for the CHO Safe-Harbor locus. Cells were transfection by electroporation using Amaxa Nucleofector 2B (Lonza) and plated in 3 mL media in a 6-well plate. After 48hrs cells were sorted by FACS and an enriched pool of the 10–15% highest labeled (GFP/Crimson) were cultured for one week, and further single cell FACS sorted and grown in 96-well plates. KO clones were identified by insertion deletion analysis (IDAA) as recently described (Yang, Steentoft et al. 2015; Lonowski et al. 2017), and when possible also screened by immunocytology with lectins and MaB. Selected clones were further verified by Sanger sequencing for characterization of exact mutations introduced. The strategy enabled fast screening and selection of KO clones with frame-shift mutations, and on average we selected 2–5 clones from each targeting event. KI clones were screened by PCR with primers specific for the junction area between the donor plasmid and the Safe-Harbor locus, and a primer set flanking the targeted KI locus was used to characterize the allelic insertion status. Immunocytology Harvested cells were air-dried overnight on Teflon slides and fixed in −20°C acetone for 7 min. Slides were stained with antihuman β4Gal-T1 mAb 2F5 (Hoffmeister et al. 2003) followed by FITC-conjugated goat antimouse (15 μg/mL; Dako), or goat antihuman ST6Gal-I antibody (1 μg/mL; cat. AF5924, R&D Systems) followed by donkey antigoat Alexa Fluor® 488-conjugated polyclonal antibody (4 μg/mL). Images were prepared on a Axioskop 2 plus microscope (Zeiss). N-glycan profiling by HILIC-UPLC N-glycans from Protein G purified IgG samples were released, enriched and labeled using Glycoworks® RapiFluro-MS kit (Waters®) according to the manufacture’s protocol (Lauber et al. 2015). In brief, approximately 15 μg of purified IgG was heated to 95°C for 3 min in buffered RapiGest solution and treated with Rapid PNGaseF at 50°C for 5 min. The sample was then cooled to room temperature and mixed with 0.8 mg RapiFluor-MS reagent. The labeling reaction time was 5 min at RT. Acetonitrile (ACN) was then added to a final concentration of 89.5% and the reaction was cleaned up using Glycoworks® HILIC μElution plate. The labeled N-glycans were subsequently analyzed on a Waters® Acquity® UPLC system equipped with a fluorescence (FLR) detector. The separation was optimized on a BEH amide column (2.1 × 150 mm2, 1.7 μm, Waters®) at 60°C with 50 mM ammonium formate, pH 4.7 as mobile phase A and 100% ACN as mobile phase B. The separation of the N-glycans was achieved with a gradient of mobile phase B decreasing from 75 to 54% in 35 min at a flow rate of 0.4 mL/min. N-glycan profiling by CE-LIF Analysis of IgG N-glycans was also performed by capillary electrophoresis (Váradi et al. 2014). Briefly, 15 μL Protein G Sepharose beads (GE Healthcare) were mixed with 200 μL conditioned medium in a 96-well plate. After washing two times with 50 mM NH4HCO3, 1 U PNGaseF (Roche) was added and the plate incubated for 1h at 50°C. The reaction mixture was then adjusted to 87.5% ACN and mixed with 15 μL pre-equilibrated carboxyl-coated magnet beads (Thermo-Scientific). Beads were washed twice in 87.5% ACN on a 96-well magnet stand, and mixed with 6 μL 40 mM APTS in 20% acetic acid and 2 μL 1 M NaBH3CN in Tetahydrofuran and incubated for 2h at 37°C. After the labeling, excess APTS was removed by washing twice in 87.5% ACN on the glycan-absorbed magnetic beads, and the labeled N-glycans were released in 40 μL MQ water. For capillary electrophoresis analysis, 2 μL of the labeled N-glycan were mixed with 8 μL HiDi Formamide and 0.05 μL 500GS-LAS standard, and injected into a genetic analyzer equipped with 24-capillary and laser-induced fluorescence detection (LIF, Thermo Fisher 3500 × l). Data were analyzed using the GeneMapper software. Supplementary data Supplementary data is available at Glycobiology online. Funding Novo Nordisk Foundation, Lundbeck Foundation, Kirsten og Freddy Johansen Fonden, Læge Sofus Carl Emil Friis og hustru Olga Doris Friis’ Legat, and the Danish National Research Foundation (DNRF107). Abbreviations ACN acetonitrile ADCC antibody-dependent-cell-cytotoxicity BACE-1 β-site APP cleaving enzyme 1 CDC complement dependent cytotoxicity CHO Chinese hamster ovary IDAA insertion deletion analysis SPPL3 signal peptide peptidase-like 3. Acknowledgements We would like to express our sincere gratitude to the SAFC Sigma team including K. Kayser and N. Sealover for their interest and help with this project. Conflict of interest statement H.C. and Z.Y. have filed patent applications with University of Copenhagen and C.K. is CEO at GlycoDisplay ApS that has obtained licenses to these patents. Author contributions M.A.S. designed, planned and performed most of the experiments and co-wrote the article. 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Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Glycobiology Oxford University Press

Glycoengineering design options for IgG1 in CHO cells using precise gene editing

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Abstract

Abstract Precise gene editing technologies are providing new opportunities to stably engineer host cells for recombinant production of therapeutic glycoproteins with different glycan structures. The glycosylation of recombinant therapeutics has long been a focus for both quality and consistency of products and for optimizing and improving pharmacokinetic properties as well as bioactivity. Structures of glycans on therapeutic glycoproteins are important for circulation, biodistribution and bioactivity. In particular, the latter has been demonstrated for therapeutic IgG1 antibodies where the core α1,6Fucose on the conserved N-glycan at Asn297 have remarkable dampening effects on antibody effector functions. We previously explored precise gene engineering and design options for N-glycosylation in CHO cells, and here we focus on engineering options possible for N-glycans on human IgG1. We demonstrate stable precise gene engineering of rather homogenous biantennary N-glycans with and without galactose (G0F, G2F) as well as the α2,6-linked monosialylated (G2FS1) glycoform. We were unable to introduce substantial disialylated glycoforms. Instead we engineered a novel monoantennary homogeneous N-glycan design with complete α2,6-linked sialic acid capping. All N-glycoforms may be engineered with and without core α1,6Fucose. The stably engineered design options enable production of human IgG antibodies with an array of distinct glycoforms for testing and selection of optimal design for different therapeutic applications. Chinese hamster ovary, galactosyltransferase, genetic engineering, mAbs, sialyltransferase Introduction Monoclonal antibodies (mAbs) constitute a growing class of recombinant glycoprotein therapeutics and currently represent about a quarter of the approved biologics in the market, and mAbs are currently in use for a variety of infectious, inflammatory, autoimmune and cancer diseases (Walsh 2014). Most therapeutic mAbs are human or humanized IgG1 glycoproteins with a conserved N-glycan at Asn297 in its Fc-region., and the structure of this N-glycan has important roles in modulating interactions with Fcγ receptors on immune cells and complement (Jefferis 2016). Processing of the Asn297 N-glycan beyond the point of first complex-type N-acetylglucosamine residues is generally incomplete. This has been shown to be due to glycan–peptide backbone interactions and steric hindrance for subsequent enzymes to efficiently process the N-glycan, and the Asn297 glycosite in IgG1 represents a classic example of site-specific N-glycostructures driven by local constraints in accessibility (Barb and Prestegard 2011). Thus, the common N-glycan structure found on recombinant expressed IgG1 is based on a biantennary N-glycan scaffold with a core α6Fucose and with low galactose and sialic acid contents (Figure 1A). The importance of the structure of the Asn297 N-glycan for effector functions of therapeutic IgG was originally demonstrated using Lec13 mutant CHO cells or rat hybridoma YB2/0 cells for production of IgG1 without the core α6Fucose, and such IgG mAbs exhibited 50-fold increased binding to FcyRIIIa and higher antibody-dependent-cell-cytotoxicity (ADCC) (Shields et al. 2002; Shinkawa et al. 2003). This discovery paved the way for several cancer therapeutic IgG1 antibodies with improved efficacy in the clinic, including Mogamulizumab (Poteligeo) and Obinutuzumab (Gazyva), which are produced in Chinese hamster ovary (CHO) cells genetically engineered to produce N-glycans with no or low fucose by knockout of the core α6fucosyltransferase Fut8 or overexpression of the bisecting β4GlcNAc-transferase MGAT3 (Umaña et al. 1999; Yamane-Ohnuki et al. 2004). Studies have further demonstrated that also galactosylation and sialylation of the Asn297 glycan are important for receptor binding and effector functions (Boyd et al. 1995; Ohmi et al. 2016; Dekkers et al. 2017), indicating that further engineering of the N-glycan structure of therapeutic IgG1 may have great biomedical potential. Fig. 1. View largeDownload slide IgG1 biantennary N-glycan engineering design options. (A) UPLC profiling analysis of N-glycans released from IgG produced in CHOWT shows a heterogeneous mixture of predominant G0F, G1F, and minor amounts of G2F. (B) KI of B4GALT1 (CHO+B4GALT1) results in almost homogeneous G2F. (C) KI of B4GALT1 and ST3GAL4 (CHO+B4GALT1/+ST3GAL4) generates heterogeneous G2F, G2FS1 and G2FS2 glycoforms. (D) KI of B4GALT1 and ST6GAL1 KI (CHO+B4GALT1/+ST6GAL1) gives mainly G2FS1 and minor G2F glycoforms. (E) B4GALT1 and ST6GAL1 KI and KO of St3gal4 and St3gal6 (CHO−St3gal4/St3gal6/+B4GALT1/+ST6GAL1) shows mainly G2FS1 and minor G2F and G2FS2 glycoforms. (F) KO of Fut8 in CHO+B4GALT1/+ST6GAL1 results in complete removal of core α1,6Fucose. Designations for monosaccharides according to the Consortium for Functional Glycomics are indicated. Designations for N-glycan structures Gal (G0–G1–G2), α6Fuc (F), and sialic acid (S1–S2). Fig. 1. View largeDownload slide IgG1 biantennary N-glycan engineering design options. (A) UPLC profiling analysis of N-glycans released from IgG produced in CHOWT shows a heterogeneous mixture of predominant G0F, G1F, and minor amounts of G2F. (B) KI of B4GALT1 (CHO+B4GALT1) results in almost homogeneous G2F. (C) KI of B4GALT1 and ST3GAL4 (CHO+B4GALT1/+ST3GAL4) generates heterogeneous G2F, G2FS1 and G2FS2 glycoforms. (D) KI of B4GALT1 and ST6GAL1 KI (CHO+B4GALT1/+ST6GAL1) gives mainly G2FS1 and minor G2F glycoforms. (E) B4GALT1 and ST6GAL1 KI and KO of St3gal4 and St3gal6 (CHO−St3gal4/St3gal6/+B4GALT1/+ST6GAL1) shows mainly G2FS1 and minor G2F and G2FS2 glycoforms. (F) KO of Fut8 in CHO+B4GALT1/+ST6GAL1 results in complete removal of core α1,6Fucose. Designations for monosaccharides according to the Consortium for Functional Glycomics are indicated. Designations for N-glycan structures Gal (G0–G1–G2), α6Fuc (F), and sialic acid (S1–S2). Genetic engineering of the glycosylation capacity of mammalian cells was for long hampered by the need for successive homologous recombination cycles to knockout unwanted endogenous glycosylation features (Yamane-Ohnuki et al. 2004). With the emergence of the precise gene editing technologies it is now possible to perform combinatorial and comprehensive KO/KI glycoengineering in mammalian cells, and develop host cell lines with a high degree of stable custom designed glycosylation capacity (Yang, Wang et al. 2015). Considerable efforts in the last decades have been devoted to engineering of the N-glycosylation capacities of mammalian, insect, yeast and plant production cells (Hamilton et al. 2006; Durocher and Butler 2009; Clausen et al. 2015), and today it is possible to produce recombinant glycoproteins in all systems with more or less heterogeneous N-glycans compatible with therapeutic use in humans (Hollister et al. 2002; Castilho et al. 2010, Laukens et al. 2015). Engineering the glycosylation capacity of cells for therapeutic IgG has, however, proved more challenging. Overexpression of galactosyltransferases and sialyltransferases in different host cells have demonstrated some increase in the galactosylation and sialylation efficiencies of the Asn297 N-glycan, but the achieved glycosylation effects have been heterogeneous and with limited degree of sialylation (Castilho et al. 2010; Dekkers et al. 2016; Lalonde and Durocher 2017). The mammalian CHO cell is by far the most used production platform for recombinant therapeutics (Walsh 2014), and the genome and endogenous N-glycosylation capacity of CHO cells are well characterized (North et al. 2010; Xu et al. 2011). It has been argued that use of nonmammalian cells with less complex glycosylation may offer better opportunities to rebuild more homogenous human N-glycosylation (Hamilton et al. 2006; Chen 2016, Kallolimath et al. 2016). However, the complex engineering required with de novo expression of entire glycosylation pathways may suffer in stability and consistency in yeast and plant systems (Martínez de Alba et al. 2013; Laukens et al. 2015). The novel opportunities with facile combinatorial precise KO/KI glycoengineering in mammalian cells clearly cement CHO as the preferred host cell line (Yang, Wang et al. 2015). Here, we systematically explored options for stable precise genetic engineering of the N-glycosylation of IgG1 in CHO cells, and we developed a design matrix that enable production of IgG with distinct N-glycan structures including novel and homogeneous monoantennary structure designs. Results The stable genetic engineering strategy We first established CHO clones stably expressing high levels (approximately 300 mg/L in batch culture) of the anti-rabies human IgG1 SO57 (Sealover et al. 2013; Yang, Wang et al. 2015). Glycoprofiling of IgG purified from these cells showed the typical N-glycan structures found on recombinant IgG expressed in CHO cells with heterogeneous G0F and minor amounts G1F and G2F and essentially undetectable nonfucosylated structures (Figure 1A). We next used these IgG expressing CHOWT clones in a combinatorial engineering strategy involving KI of human glycosyltransferase genes to improve galactosylation and alpha2,3sialylation, as well as to introduce the more predominant human α2,6sialylation. We also used KO to disrupt endogenous glycosyltransferase genes in order to reduce heterogeneity and eliminate core fucosylation and biantennary glycans. All KO events were verified by gene sequencing (Supplementary data, Table S1) and unaltered expression of IgG was verified by ELISA. To circumvent stability problems with random plasmid integration, we first used a modified ObLiGaRe strategy (Maresca et al. 2013; Yang, Wang et al. 2015), for targeted KI of codon optimized full coding constructs of human genes B4GALT1, ST3GAL4 and ST6GAL1 into a Safe-Harbor locus, without use of antibiotic selection. The Safe-Harbor locus was recently identified and demonstrated to enable stable transcription during cell culture processes without affecting expression of adjacent genes (Bahr et al. 2016). We designed the donor plasmids for targeted KI with two flanking ZFN binding sites to ensure clean targeting without introducing any backbone sequence of donor plasmid. Furthermore, we included two tDNA insulator elements flanking the transcription unit to minimize epigenetic silencing (Gaszner and Felsenfeld 2006). In order to stack multiple exogenous genes by KI, we applied an alternative strategy using PCR amplicon of the constructs described above together with CRISPR-Cas9 for targeted KI of a second gene into the Safe-Harbor gRNA recognition locus, localized adjacent to the ZFN binding site. KI clones were initially screened by immunocytology with antibodies and lectins (Supplementary data, Figure S1), and correct targeted integration into the Safe-Harbor site was confirmed by junction PCR (Supplementary data, Figure S2). Interestingly, the first screening by immunocytology showed variable and heterogeneous Golgi-like staining in the initial pool of cells, and clones with homogenous and strong staining were selected and found to maintain stable expression of the KI glycosyltransferase genes. Engineering biantennary N-glycoforms on human IgG1 We previously showed that KO of the endogenous B4galt1 galactosyltransferase gene in CHO cells resulted in production of IgG with homogeneous G0F N-glycans (Yang, Wang et al. 2015), and here we found that monoallelic targeted KI of human B4GALT1 (CHO+B4GALT1) resulted in significantly improved galactosylation of IgG and essentially a homogeneous G2F glycoform with very limited sialylation (Figure 1B). To introduce sialylation, we tested KI of human ST3GAL4 for α2,3sialylation and ST6GAL1 for α2,6sialylation, as these have been shown to be the most efficient sialyltransferase isoenzymes acting on N-glycans in CHO (Yang, Wang et al. 2015). Targeted KI of ST3GAL4 into CHO+B4GALT1 to generate CHO+B4GALT1/+ST3GAL4 did improve α2,3sialylation but only partially (Figure 1C). In contrast, monoallelic KI of ST6GAL1 into CHO+B4GALT1 to generate a CHO+B4GALT1/+ST6GAL1 resulted in conversion of the G2F glycoform to a rather homogenous G2FS1 glycoform with minor amounts of G2F and detectable G2FS2 (Figure 1D). To explore potential interference from endogenous α2,3sialyltransferases and ensure that sialylation was homogeneously α2,6-linked, we further stacked KO of the two predominant endogenous sialyltransferase genes, St3gal4 and St3gal6, responsible for sialylation of N-glycans in CHO (Yang, Wang et al. 2015). This did not appear to affect the degree of sialylation in the CHO+B4GALT1/+ST6GAL1 cell line (Figure 1E). The core α6Fucose on N-glycans is controlled by the Fut8 gene, and KO of this gene results in complete loss of α6Fuc (Miyoshi et al. 1999; Yang, Wang et al. 2015). We confirmed this in the CHO+B4GALT1/+ST6GAL1 line by stacked KO of Fut8 (Figure 1F). The KO of Fut8 appeared to lower the efficiency of sialylation slightly, but it is unclear if this is related to the clonal ancestry or it is a general feature. Clearly further studies are needed, but we predict that any of the presented N-glycan designs are largely interchangeable with and without the core Fucose. Engineering monoantennary N-glycoforms on human IgG1 Inspired by our previous studies engineering monoantennary N-glycoforms (Yang, Wang et al. 2015), as well as the GlycoDelete concept (Meuris et al. 2014), we explored monoantennary design options for the conserved N-glycan on IgG by KO of the Mgat2 gene directing biantennary formation (Figure 2A). KO of Mgat2 in CHOWT produced a heterogeneous mixture of monoantennary N-glycans with and without galactosylation and sialylation (G0F, G1F, G1FS1) (Figure 2B). KO of Mgat2 in the B4GALT1 monoallelic KI clone CHO+B4GALT1 to generate CHO−Mgat2/+B4GALT1 resulted in monoantennary N-glycans with complete galactosylation, and interestingly the endogenous α2,3sialylation remained incomplete to generate a mix of monoantennary G1F and G1FS1 (Figure 2C). We further stacked ST3GAL4 by targeted KI into CHO−Mgat2/+B4GALT1 to generate CHO−Mgat2/+B4GALT1/+ST3GAL4, which presented a marked increase in α2,3sialylation, although some nonsialylated G1F remained (Figure 2D). Removal of the endogenous sialylation by KO of St3gal4 and St3gal6 in CHO−Mgat2/+B4GALT1 (CHO−Mgat2/−St3gal4/−St3gal6/+B4GALT1), resulted in homogenous monoantennary N-glycans devoid of sialylation but with complete galactosylation (G1F) (Figure 2E). To obtain homogenous α2,6sialylation, we did targeted KO of St3gal4 and St3gal6 to eliminate α2,3sialylation capacity along with Mgat2 KO into the double KI clone CHO+B4GALT1/+ST6GAL1 (CHO−Mgat2/−St3gal4/−St3gal6/+B4GALT1/+ST6GAL1), which resulted in efficient α2,6sialylation capacity and a homogeneous G1FS1 glycoform (Figure 2F). Fig. 2. View largeDownload slide IgG1 monoantennary N-glycan engineering design options. (A) UPLC profiling analysis of N-glycans released from IgG produced in CHOWT. (B) KO of Mgat2 in CHOWT generates heterogeneous monoantennary G0F, G1F and G1FS1 (CHO−Mgat2). (C) Additional KI of B4GALT1 (CHO−Mgat2/+B4GALT1) results in G1F and G1FS1. (D) Further KI of ST3GAL4 (CHO−Mgat2/+B4GALT1/+ST3GAL4) shows an increase in G1FS1 with 2,3-linked sialic acid but remaining G1F. (E) KO of St3gal4 and St3gal6 in CHO−Mgat2/+B4GALT1 results in homogeneous G1F and (F) additional KI of ST6GAL1 (CHO−Mgat2/−St3gal4/−St3gal6/+B4GALT1/+ST6GAL1) generates homogeneous G1FS1 with 2,6-linked sialic acid. (G) KO of Man2A1 and Man2A2 in CHOWT introduces an alternative monoantennary design with a tri-mannose core on the α6-arm. All glycoforms are predicted to convert to nonfucosylated with KO of Fut8. Fig. 2. View largeDownload slide IgG1 monoantennary N-glycan engineering design options. (A) UPLC profiling analysis of N-glycans released from IgG produced in CHOWT. (B) KO of Mgat2 in CHOWT generates heterogeneous monoantennary G0F, G1F and G1FS1 (CHO−Mgat2). (C) Additional KI of B4GALT1 (CHO−Mgat2/+B4GALT1) results in G1F and G1FS1. (D) Further KI of ST3GAL4 (CHO−Mgat2/+B4GALT1/+ST3GAL4) shows an increase in G1FS1 with 2,3-linked sialic acid but remaining G1F. (E) KO of St3gal4 and St3gal6 in CHO−Mgat2/+B4GALT1 results in homogeneous G1F and (F) additional KI of ST6GAL1 (CHO−Mgat2/−St3gal4/−St3gal6/+B4GALT1/+ST6GAL1) generates homogeneous G1FS1 with 2,6-linked sialic acid. (G) KO of Man2A1 and Man2A2 in CHOWT introduces an alternative monoantennary design with a tri-mannose core on the α6-arm. All glycoforms are predicted to convert to nonfucosylated with KO of Fut8. An alternative approach to generate a monoantennary N-glycan structure was achieved by KO of the mannosidase genes, Man2A1 and Man2A2, in CHOWT (Figure 2G). This resulted in a rather heterogeneous monoantennary structure with an extended mannose structure on the α6-arm and a partially galactosylated and sialylated α3-arm. We predict that further combined KI of B4GALT1 and ST6GAL1 and KO of St3gal4 and St3gal6 would result in a homogeneous monoantennary structure as demonstrated for the engineering sequence based on the Mgat2 KO, but with a more elaborate α6-arm. Targeting ER/Golgi proteins known to effect transport and residency of glycosyltransferases Studies have demonstrated that the stability of Golgi glycosyltransferases, including β4Gal-T1 and ST6Gal-I, may be regulated by resident proteases such as signal peptide peptidase-like 3 (SPPL3) (Voss et al. 2014; Kuhn et al. 2015). We therefore investigated if targeted KO of this protease would improve glycosylation efficiencies in CHO cells. KO of the Sppl3 gene in CHOWT cells appeared to increase galactosylation of IgG1 slightly, however, KO of Sppl3 in the CHO+B4GALT1 and CHO+B4GALT1/+ST6GAL1 KI clones did not substantially affect the glycosylation profile or improve α2,6 sialylation. Similarly, β-site APP cleaving enzyme 1 (BACE-1) has previously been shown to cleave ST6Gal-I (Kitazume et al. 2003), but KO of the Bace1 gene did not appear to affect the sialylation efficiency in the CHO+B4GALT1/+ST6GAL1 clone (not shown), and we therefore did not pursue these engineering strategies further. Evaluation of clonal stability In agreement with our previous study, we found that CHO cells have great plasticity for targeted KO/KI glycoengineering (Yang, Wang et al. 2015), as we did not observe gross effects on viability, growth, and recombinant production of the IgG for any of the genetic designs introduced (Figure 3A). To evaluate this in more detail, we chose the CHO+B4GALT1/+ST6GAL1 double KI cell line and cultured it for 25 passages, which represents at least 50 generations and is beyond the needs for full scale up of antibody production cell lines. The IgG N-glycosylation was identical at passages 1 and 25 (Figure 3B). Fig. 3. View largeDownload slide Stability of glycodesigns. (A) Growth and viability analysis over 8 days for CHOWT and CHO+B4GALT1 clones. The CHO clones were seeded at 0.25 million cells/mL in 50 mL shaking tubes with 30 mL media volume. Mean of three independent cultures. (B) Glycoprofiling by CE-LIF of a CHO+B4GALT1/+ST6GAL1 clone before and after growth over 25 passages in 50 mL shake tubes. Fig. 3. View largeDownload slide Stability of glycodesigns. (A) Growth and viability analysis over 8 days for CHOWT and CHO+B4GALT1 clones. The CHO clones were seeded at 0.25 million cells/mL in 50 mL shaking tubes with 30 mL media volume. Mean of three independent cultures. (B) Glycoprofiling by CE-LIF of a CHO+B4GALT1/+ST6GAL1 clone before and after growth over 25 passages in 50 mL shake tubes. Discussion Here, we continued our glycoengineering efforts using precise gene editing to establish stable CHO cell lines with well-defined N-glycosylation capacities designed specifically for production of therapeutic IgGs. The design matrix presented for stable glycoengineering of CHO cells enables production of IgG antibodies with traditional biantennary G0, G2, G2S1 with and without core fucose. We were unable to introduce the homogeneous disialylated G2FS2 glycoform, and this glycoform has thus far only been achieved by extensive in vitro enzymatic glycosylation (Huang et al. 2012; Washburn et al. 2015). The G2FS2 glycoform has also been produced chemoenzymatically using a mutagenized endoglucosidase (EndoS) with transglycosylation activity to introduce a synthetic biantennary glycans (Huang et al. 2012). A number of studies have shown that the biological activity of IgG1 is influenced by the structure of the Asn297 N-glycan in the Fc region (Wuhrer et al. 2015; Jefferis 2016; Lu et al. 2016). Other N-glycan features influence the flexibility of the Fc domain and affect the interactions with immune receptors. Thus, increased galactosylation improves C1q binding and complement dependent cytotoxicity (CDC) (Dekkers et al. 2017; Peschke et al. 2017), while α2,6sialylation abrogates this effect (Quast et al. 2015). Increased galactosylation has been shown to improve IgG binding to FcγRIIIa and increase ADCC (Thomann et al. 2015), while effects of α2,6sialylation on FcγRIIIa binding is more controversial (Thomann et al. 2015; Lin, Tsai et al. 2015; Dekkers et al. 2017). Increased 2,6-sialylation of intravenous immunoglobulin and IgGs has been shown to induce an anti-inflammatory activity (Kaneko et al. 2006; Anthony et al. 2008; Oefner et al. 2012, Ohmi et al. 2016; Pagan et al. 2018). Although our CHO design matrix allows stable and homogeneous productions of IgG1 to further investigate glycan dependent biological activities, circulating IgG may undergo extracellular sialylation by secreted ST6Gal1 (Jones et al. 2016) and is a possible additional level of regulation of the immunomodulatory functions of IgG. Various strategies have been pursued to produce IgG with different glycoforms. Culture conditions may improve glycosylation efficiencies and especially sialylation slightly, but heterogeneity always remains (Jefferis 2016). The first genetic glycoengineering in CHO cells introduced α2,6sialylation capacity by stable plasmid transfection with ST6GAL1 (Lee et al. 1989), and since then a large number of studies have used overexpression of α2,3- or α2,6-sialyltransferases with or without β4-galactosyltransferases in CHO (Weikert et al. 1999; El Maï et al. 2013; Lin, Mascarenhas et al. 2015; Raymond et al. 2015). Heterogeneity and incomplete galactosylation and sialylation were consistent issues, although one study suggests that overexpression of B4galt1 and St6gal1 in a murine hybridoma cell line may lead to the fully sialylated G2FS2 N-glycan structure on mouse IgG1 (Ohmi et al. 2016). In agreement with these studies, we were able to induce efficient galactosylation (G2F) and sialylation (G2FS1), although significant levels of G2FS2 were not reached, stressing the difficulties with glycosylation of the Asn297 N-glycan (Figure 1). Stable genetic engineering of the glycosylation capacities of mammalian cells have only recently been facilitated by precise gene editing technologies (ZFNs, TALENs and CRISPR/Cas9) (Steentoft et al. 2014). Eliminating unwanted glycosylation capacities is fairly straightforward using KO of individual glycogenes, and stacking multiple targeting events in CHO cells is also now possible (Yang, Wang et al. 2015). Moreover, targeted KI into Safe-Harbor sites enables selection of stable glycosyltransferase KI clones with homogeneous expression levels (Figure 1). Once selected the enzyme expression and glycan profile were stable and consistent beyond the cell generations generally required for industrial scaled fed-batch production (Figure 3). The precise gene engineering enabled us to establish cell lines producing a level of homogeneity in glycosylation of IgG previously unreachable (Figure 1). An elegant example of an entirely novel glycoengineering strategy for IgG1 is presented with GlycoDelete (Meuris et al. 2014), where a Golgi-targeted fungal endoglucosidase (endoT) is expressed in human HEK293 cells to generate simple N-glycans with a heterogeneous mix of mono-, di- or trisaccharide structures (Neu5Ac–Gal–GlcNAc-Asn) resulting in a relative homogeneous Fc-region on an anti-CD20 mAb. Interestingly, the GlycoDelete antibody showed less initial clearance from serum in mice, and a lower binding affinity to human Fcγ receptors. The long circulatory half-life of IgG1 of 3 weeks is largely governed by cellular recycling by the neonatal Fc-receptor (FcRn) that binds independent of the N-glycan (Tao and Morrison 1989; Burmeister et al. 1994). This imply that glycosylation, and in particular sialylation, may not be relevant for circulation in contrast to most other therapeutic glycoproteins (Fukuda et al. 1989). However, truncating the N-glycan by GlycoDelete appears to improve initial circulation somewhat (Meuris et al. 2014). Here, we pursued a different strategy to generate simpler and homogeneous N-glycans on IgG with our monoantennary design (Figure 2), and future studies are needed to explore the biological consequences of these designs. In summary, we presented specific engineering options for the unique N-glycan at Asn297 on human IgG1. While we were unable to engineer the biantennary G2FS2 N-glycoform, we obtained CHO cells with rather homogeneous G2F and G2FS1 glycoforms, as well as novel homogeneous monoantennary G1F and G1FS1 glycoforms. Availability of the design matrix and engineered cells for consistent production of different human IgG1 glycoforms will now enable wider studies and productions of optimal glycoforms for specific therapeutic applications. Materials and methods Recombinant expression of IgG in CHO cells The anti-rabies human IgG1 SO57 (Sealover et al. 2013) was used to establish stable expressing CHO clones. Briefly, we constructed and transfected a pEE-IgG vector (Lonza Biologics) into CHOZN GS−/− cells (Sigma-Aldrich) before glutamine selection was used to select stable IgG expressing clones. All media, supplements and other reagents were obtained from Sigma-Aldrich unless otherwise specified. CHO cells were maintained as suspension cultures in EX-CELL CHO CD Fusion serum-free media, supplemented with 4 mM l-glutamine either in 50 mL TPP TubeSpin® Bioreactors at 200 rpm or 100 mL in Corning 500 mL culture bottle at 130 rpm (Infors), both at 36.5°C and 5% CO2 in air. For IgG production, clones were seeded at 0.5 × 106 cells/mL without l-glutamine and cultured for 3 days for IgG production, and IgG was purified by HiTrapTM Protein G HP (GE Healthcare, US) pre-equilibrated in PBS and eluted with 0.1 M glycine (pH 2.7). Purity of proteins was evaluated by Coomassie staining of SDS-PAGE gels, and proteins were quantified by BCA Protein Assay Kit (Thermo Scientific) or direct ELISA. For stability studies cells were grown in 50 mL tubes with 30 mL media volume, and split (1:8 ratio) two times every week with seeding at 0.25 million cells/mL. Gene targeting in CHO cells Gene targeting by CRISPR/Cas9 and ZFN were performed in CHO cells expressing IgG and 25–100 clones were screened for each targeting event. Cells were seeded at 0.5 × 106 cells/mL 2 days prior to transfection. KO was performed with 1 μg endotoxin-free plasmid DNA of each Cas9-GFP and gRNA in px458 vector (Addgene). KI was performed with 2 μg of each ZFN tagged with GFP/Crimson as previously described (Duda et al. 2014), and 5 μg donor plasmid with full coding codon-optimized human glycosyltransferase genes (B4GALT1, ST3GAL4 or ST6GAL1). To KI a second gene into the Safe-Harbor locus we used 1 μg of a PCR product encoding the codon optimized human enzymes with 1 μg Cas9-GFP and gRNA for the CHO Safe-Harbor locus. Cells were transfection by electroporation using Amaxa Nucleofector 2B (Lonza) and plated in 3 mL media in a 6-well plate. After 48hrs cells were sorted by FACS and an enriched pool of the 10–15% highest labeled (GFP/Crimson) were cultured for one week, and further single cell FACS sorted and grown in 96-well plates. KO clones were identified by insertion deletion analysis (IDAA) as recently described (Yang, Steentoft et al. 2015; Lonowski et al. 2017), and when possible also screened by immunocytology with lectins and MaB. Selected clones were further verified by Sanger sequencing for characterization of exact mutations introduced. The strategy enabled fast screening and selection of KO clones with frame-shift mutations, and on average we selected 2–5 clones from each targeting event. KI clones were screened by PCR with primers specific for the junction area between the donor plasmid and the Safe-Harbor locus, and a primer set flanking the targeted KI locus was used to characterize the allelic insertion status. Immunocytology Harvested cells were air-dried overnight on Teflon slides and fixed in −20°C acetone for 7 min. Slides were stained with antihuman β4Gal-T1 mAb 2F5 (Hoffmeister et al. 2003) followed by FITC-conjugated goat antimouse (15 μg/mL; Dako), or goat antihuman ST6Gal-I antibody (1 μg/mL; cat. AF5924, R&D Systems) followed by donkey antigoat Alexa Fluor® 488-conjugated polyclonal antibody (4 μg/mL). Images were prepared on a Axioskop 2 plus microscope (Zeiss). N-glycan profiling by HILIC-UPLC N-glycans from Protein G purified IgG samples were released, enriched and labeled using Glycoworks® RapiFluro-MS kit (Waters®) according to the manufacture’s protocol (Lauber et al. 2015). In brief, approximately 15 μg of purified IgG was heated to 95°C for 3 min in buffered RapiGest solution and treated with Rapid PNGaseF at 50°C for 5 min. The sample was then cooled to room temperature and mixed with 0.8 mg RapiFluor-MS reagent. The labeling reaction time was 5 min at RT. Acetonitrile (ACN) was then added to a final concentration of 89.5% and the reaction was cleaned up using Glycoworks® HILIC μElution plate. The labeled N-glycans were subsequently analyzed on a Waters® Acquity® UPLC system equipped with a fluorescence (FLR) detector. The separation was optimized on a BEH amide column (2.1 × 150 mm2, 1.7 μm, Waters®) at 60°C with 50 mM ammonium formate, pH 4.7 as mobile phase A and 100% ACN as mobile phase B. The separation of the N-glycans was achieved with a gradient of mobile phase B decreasing from 75 to 54% in 35 min at a flow rate of 0.4 mL/min. N-glycan profiling by CE-LIF Analysis of IgG N-glycans was also performed by capillary electrophoresis (Váradi et al. 2014). Briefly, 15 μL Protein G Sepharose beads (GE Healthcare) were mixed with 200 μL conditioned medium in a 96-well plate. After washing two times with 50 mM NH4HCO3, 1 U PNGaseF (Roche) was added and the plate incubated for 1h at 50°C. The reaction mixture was then adjusted to 87.5% ACN and mixed with 15 μL pre-equilibrated carboxyl-coated magnet beads (Thermo-Scientific). Beads were washed twice in 87.5% ACN on a 96-well magnet stand, and mixed with 6 μL 40 mM APTS in 20% acetic acid and 2 μL 1 M NaBH3CN in Tetahydrofuran and incubated for 2h at 37°C. After the labeling, excess APTS was removed by washing twice in 87.5% ACN on the glycan-absorbed magnetic beads, and the labeled N-glycans were released in 40 μL MQ water. For capillary electrophoresis analysis, 2 μL of the labeled N-glycan were mixed with 8 μL HiDi Formamide and 0.05 μL 500GS-LAS standard, and injected into a genetic analyzer equipped with 24-capillary and laser-induced fluorescence detection (LIF, Thermo Fisher 3500 × l). Data were analyzed using the GeneMapper software. Supplementary data Supplementary data is available at Glycobiology online. Funding Novo Nordisk Foundation, Lundbeck Foundation, Kirsten og Freddy Johansen Fonden, Læge Sofus Carl Emil Friis og hustru Olga Doris Friis’ Legat, and the Danish National Research Foundation (DNRF107). Abbreviations ACN acetonitrile ADCC antibody-dependent-cell-cytotoxicity BACE-1 β-site APP cleaving enzyme 1 CDC complement dependent cytotoxicity CHO Chinese hamster ovary IDAA insertion deletion analysis SPPL3 signal peptide peptidase-like 3. Acknowledgements We would like to express our sincere gratitude to the SAFC Sigma team including K. Kayser and N. Sealover for their interest and help with this project. Conflict of interest statement H.C. and Z.Y. have filed patent applications with University of Copenhagen and C.K. is CEO at GlycoDisplay ApS that has obtained licenses to these patents. Author contributions M.A.S. designed, planned and performed most of the experiments and co-wrote the article. 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Google Scholar CrossRef Search ADS PubMed © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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GlycobiologyOxford University Press

Published: Mar 27, 2018

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