Three constitutive short hairpin siRNA expression vectors against Chinese hamster FUT8, GMD, and GFT were generated and introduced into an IgG1 antibody-producing CHO/DG44 clone, 32-05-12 26, to evaluate the effects of target gene knockdown on the levels of fucosylation of the products. Puromycin-resistant clones transformed by the vectors appeared after transfection with a transformation efficiency of approximately 1,300 per 1.6 × 10⁶ electroporated cells, irrespective of the vector differences. However, subsequent selection of LCA-resistant clones showed clear differences in the appearance of surviving colonies. The ratios of LCA-resistant clones to puromycin-resistant clones transformed by the three siRNA expression vectors against FUT8, GMD, and GFT were 11.5%, 14.8%, and 0.8%, respectively (Table 1). The resultant LCA-resistant clones were expanded in medium without LCA, and the mRNA expression of FUT8, GMD, and GFT, as well as the oligosaccharide structure of the antibodies produced in these clones, were quantitatively analyzed. The target gene mRNA was reduced to a level exceeding 90% in clones appearing in all three siRNA-introduced transformants; however, in none of the clones was Fc oligosaccharide fucosylation completely abolished. The most effective rates of reduction in fucosylation among the clones transformed by the FUT8, GMD, and GFT siRNA expression vectors were approximately 72%, 79%, and 42%, respectively.

To explore the effects of double knockdown of the target genes FUT8 and GMD, two GMD siRNA-introduced clones exhibiting a maximum reduction in product fucosylation, designated as GMDb2 and GMDb5, were selected and re-electroporated with the FUT8 siRNA expression vector. LCA-resistant clones were selected in the presence of 100 μM L-fucose after drug-resistant selection had been carried out, and two clones expressing highly non-fucosylated antibodies were selected independently from each GMD siRNA-introduced clone in the medium. In the four established clones, the FUT8 mRNA expression levels decreased to roughly one-tenth of the parental expression levels, while the original 90% decrease in GMD mRNA was retained. Monosaccharide composition analysis showed that the ratio of non-fucosylated oligosaccharides of the antibodies produced by the four clones had increased significantly in each case (i.e., up to 91%, 95%, 97%, and 98%), compared to those of parental GMD siRNA-introduced clones (Table 2). The increase in non-fucosylated product levels was cancelled when the clones were cultured in the presence of L-fucose; this cancellation was ascribed to the absence of a GMD knockdown effect due to activation of the salvage pathway of GDP-fucose synthesis. No significant effect on fucosylation levels in antibody-producing cells was observed with a combination of either FUT8 and GFT or GMD and GFT double knockdown.

To facilitate the double knockdown of FUT8 and GMD, the siRNA tandem expression vector for targeting these genes was generated and introduced into IgG1 antibody-producing CHO/DG44 32-05-12 cells. Hygromycin-resistant clones transformed by the vectors appeared with a common transformation efficiency of approximately 1,500 per 1.6 × 10⁶ electroporated cells. However, subsequent selection of LCA-resistant clones revealed clear differences in the appearance of surviving colonies, as compared to that of single siRNA-introduced clones (Table 1). The ratio of LCA-resistant clones to hygromycin-resistant clones transformed by the siRNA tandem expression vector was much higher (more than triple) than that of clones transformed by the vector bearing each single siRNA expression cassette; approximately half of the hygromycin-resistant clones showed resistance to LCA, even in the culture condition containing L-fucose. Among the resultant LCA-resistant clones, twenty were randomly selected and expanded in medium lacking both LCA and L-fucose. The mRNA expression of both target genes, FUT8 and GMD, in each clone decreased to approximately less than 20% of that of the parental clone (Fig. 2A). Out of twenty clones, eight exhibited relatively low levels of both target genes, and these were selected for further analysis of the oligosaccharide structure of the antibody products. The Fc oligosaccharide structures of the antibodies produced by these eight clones showed almost complete non-fucosylation; there were no detectable L-fucose residues among the products from six clones (Table 3). These six clones exhibited very low reactivity to LCA, even after adaptation to serum-free medium, as was also observed in the case of the FUT8-knockout CHO cell line Ms705. The results obtained from two representative clones, designated as FG1 and FG16, are shown in Fig. 2B–2E.

Serum-free fed-batch culture of the clones transformed by the FUT8 and GMD siRNA tandem expression vector was carried out using 1L-scale spinner bioreactors with pH and DO controls. Two FUT8 and GMD siRNA-introduced clones, FG1 and FG16, and their parental IgG1-producing clone, CHO/DG44 32-05-12, were adapted to serum-free medium, and the performance of the fed-batch cultures was compared in a head-to-head analysis (Fig. 3A, 3B). The fed-batch cultures were maintained until the cell viability decreased to less than 50%, which occurred at day 16 post-inoculation. Culture aliquots were taken at days 3, 6, 9, 12, 14, and 16 to analyze the cells and antibody products. Two siRNA-introduced cell lines grew logarithmically, with a slight difference in the specific production rate, maximum viable cell density, and the day upon which the viable cell density reached the maximum level; two siRNA-introduced hygromycin-resistant descendants showed slightly slower growth and death rates than those of the parent cells. However, the productivity of two clones transformed by FUT8 and GMD siRNAs reached a production level of approximately 1200 mg/L, which was basically equivalent to that of the parental cells at the end of the fed-batch culture period. During the culture period, the levels of cellular FUT8 and GMD mRNAs in the two siRNA-introduced clones remained at the original low level, i.e., less than 20% of the parental level. Monosaccharide composition analysis revealed that the two siRNA-introduced clones stably produced non-fucosylated antibodies throughout the culture period (Table 4). The oligosaccharide profile analysis of products purified from the final culture medium confirmed that the N-linked Fc oligosaccharides of the products were of the biantennary complex type, and the products from the two siRNA-introduced clones fully lacked core fucosylation (Fig. 3D, 3E). It was also confirmed that there was no significant change in the oligosaccharide profile during the culture period, and the non-fucosylated products showed two orders of magnitude higher ADCC than the antibodies from the parental CHO/DG44 cells, without any changes in antigen binding (Fig. 4).

The dihydrofolate reductase-deficient CHO cell line, CHO/DG44 36, was obtained from Dr. Lawrence Chasin of Columbia University (New York). A recombinant mouse/human chimeric IgG1-producing CHO/DG44 cell line, 32-05-12, generated as described previously 26, was cultured in IMDM medium (Invitrogen, Carlsbad, CA) containing 10% (v/v) dialyzed fetal bovine serum (dFBS; Invitrogen) and 500 nM methotrexate (MTX; Sigma-Aldrich, St. Louis, MO).

An siRNA expression plasmid for targeting Chinese hamster GMD (GenBank: AF525364), pPUR/GMDshB, was generated as described below (Fig. 5A). The plasmid contained a puromycin resistance gene as a selection marker and a GMD short hairpin siRNA expression cassette controlled by the human U6 promoter. The fragment of human U6 promoter was prepared by PCR with KOD polymerase (TOYOBO, Tokyo, Japan) from the plasmid U6_FUT8_B_puro 26 using the primers 5'-CCCAAGCTTG ATATCAAGGT CGGGCAGGAA GAGGGCCTAT-3' and 5'-GCTCTAGAGA TATCAAAAAA GGTACCGAGC TCGGTGTTTC GTCCTTTCCA CA-3'. The amplified human U6 promoter was inserted into pPUR (Clontech, Mountain View, CA) at the PvuII site, and the synthetic dsDNA coding short hairpin siRNA against GMD (5'-GAGCTC

TATA AGAATCCACA GGCTCATATT GAAGG

CCTTC AATATGAGCC TGTGGATTCT TATA

An siRNA expression plasmid for targeting Chinese hamster FUT8 28, FUT8shRNA/lib3/pPUR, contained a puromycin resistance gene as a selection marker and a FUT8 short hairpin siRNA expression cassette controlled by the human tRNA^val promoter (Fig. 5B). The fragment of human tRNA^val promoter was prepared by PCR from the plasmid ptRNA-SS 37 using the primers 5'-TTCCCAGTCA CGACGTT-3' and 5'-CAGGAAACAG CTATGAC-3'. The amplified human tRNA^val promoter was inserted into pPUR using the PvuII site, and the synthetic dsDNA coding short hairpin siRNA against FUT8 (5'-GAGCTC

AAAT CCAAAAGAAT TTCATCTGCA TGTCTTTGG

CCAAA GACATGCAGA TGAAATTCTT TTGGATTT

Another FUT8 siRNA expression plasmid, FT8lib3/pAGE (Fig. 5C), was constructed by insertion of the FUT8 short hairpin siRNA expression cassette from FUT8shRNA/lib3/pPUR into pAGE249 8 as follows. The FUT8 short hairpin siRNA expression cassette was excised from FUT8shRNA/lib3/pPUR using the EcoRI and XhoI sites, and its EcoRI terminus was converted to SmaI by subcloning into pBlusecriptII KS(+) (Stratagene, La Jolla, CA). The SmaI-XhoI fragment containing the FUT8 short hairpin siRNA expression cassette was inserted into pAGE249 at the NaeI and XhoI sites to construct FT8lib3/pAGE.

Two siRNA expression plasmids for targeting Chinese hamster GFT (GenBank: AB222037), GFT_3G10/pPUR and GFT_3G10/pAGE, were constructed by replacement of the FUT8 dsDNA of FUT8shRNA/lib3/pPUR and FT8lib3/pAGE, respectively, with the dsDNA coding short hairpin siRNA against GFT (5'-GAGCTC

CCAA AGAGGGTGAG AAGAGTGCTA TTG

CAATAGCACTC TTCTCACCCT CTTTGG

Finally, an siRNA tandem expression plasmid for the double knockdown of FUT8 and GMD, FT8lib3_GMDB/pAGE, was constructed by insertion of the GMD short hairpin siRNA expression cassette (amplified by PCR from pPUR/GMDshB using the primers 5'-CCGCTCGAGA GCGCCTGATG CGGTATT-3' and 5'-CCGCTCGAGG GACTTTCCAC ACCTGGT-3') into FT8lib3/pAGE at the XhoI site (Fig. 5D). In this siRNA tandem expression vector, different PolIII promoters, tRNA^val and U6, were designed to prevent unnecessary interference between the two promoter systems.

In order to quantify the amounts of GMD, FUT8, and GFT mRNA in the cells, real-time PCR analysis was carried out using TaKaRa Ex Taq™ R-PCR Version (TaKaRa, Shiga, Japan) and the following four sets of primers: 5'-ATCCTCGTCC TCCTTACTTA CC-3' and 5'-TCCAGCTGAC CAAGAAATAG AG-3' for FUT8, 5'-AAGCCCAGGA AGGTGGCGCT CATCAC-3'and 5'-CACTAGTTGA GGCCTGGTAG AACTTCAC-3' for GMD, 5'-ATCATCATTG GTGGTTTCTG G-3' and 5'-TCTCTTCATA GTAGAGCACG GC-3' for GFT, and 5'-GATATCGCTG CGCTCGTCGT CGAC-3' and 5'-CAGGAAGGAA GGCTGGAAGA GAGC-3' for β-actin as an internal standard gene. Total RNA was isolated from 5 × 10⁶ cells using an RNeasy minikit (Qiagen, Hilden, Germany). Single-strand cDNA was synthesized from 3 μg total RNA using the Superscript™ III first-strand synthesis system for RT-PCR (Invitrogen). A 50-fold diluted reaction mixture was used as a template. PCR was carried out by heating the mixture at 94°C for 5 min followed by 40 cycles of 94°C for 20 s, 65°C for 1 min, and 72°C for 30 s in 20 μL of reaction mixture containing 1 unit of TaKaRa Ex Taq™ R-PCR Version, 1 μL of 2500-fold diluted SYBR Green I (TaKaRa), 5 μL of the diluted single-strand cDNA, and 6 pmol of primers using an ABI PRISM 7700 sequence detection system (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. After PCR amplification, data acquisition and analyses were performed using the GeneAmp 7700 sequence detection system version 1.7 (Applied Biosystems).

Cells (2 × 10⁵) were suspended in phosphate buffered saline (PBS) containing 1% bovine serum albumin (BSA), and either 2 μg/mL fluorescein isothiocyanate (FITC)-labeled LCA (Vector Laboratories, Burlingame, CA) or 2 μg/mL FITC-labeled streptavidin (KPL, Gaithersburg, MD) were added to the suspension. After incubation at 4°C for 30 min, 1 × 10⁴ stained cells were analyzed by FACScalibur (BD Biosciences, San Jose, CA) to evaluate the reactivity to LCA on the cell surface.

Recombinant antibodies were purified from the serum-free culture supernatant by Protein A-affinity chromatography using MabSelect™ (Amersham Biosciences, Piscataway, NJ) and the samples were stored in 10 mM KH₂PO₄. The concentration of purified antibodies was measured by absorbance at 280 nm. The monosaccharide composition of each purified IgG1 was characterized by modified high-performance anion exchange chromatography (HPAEC) as previously described 8. The oligosaccharide profile of each purified antibody was characterized by modified matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in positive-ion mode, as described previously 29.

An ADCC assay for each purified IgG1 was performed by lactate dehydrogenase (LDH) release assay using human peripheral blood mononuclear cells (PBMCs) from healthy donors as effector cells at an E:T ratio of 20:1, as described previously 26. The antigen-binding activities of the antibodies were measured by antigen-binding ELISA, as previously described 26.

An IgG1 antibody-producing clone, CHO/DG44 32-05-12 (1.6 × 10⁶ cells), was transformed by electroporation with 10 μg of each siRNA expression vector linearized at the FspI site (pPUR/GMDshB, FUT8shRNA/lib3/pPUR, or GFT_3G10/pPUR). Transfectants were selected in 12 μg/mL puromycin (Sigma-Aldrich) for 7 days, and then the drug-resistant clones were subjected to 7-day selection with 500 μg/mL LCA. The resultant LCA-resistant clones were isolated and expanded in IMDM medium containing 10% (v/v) dFBS, 500 nM MTX, and 12 μg/mL puromycin. Cells were collected for real-time PCR analysis after they had grown to confluence in a tissue-culture flask (Greiner, Frickenhausen, Germany). The serum-free culture supernatants were recovered for antibody analysis after a 7-day culture of the confluent cells in serum-free EX-CELL™ 301 medium (JRH Biosciences, Lenexa, KS).

The isolated GMD siRNA-introduced clones (1.6 × 10⁶ cells) were electroporated again with 10 μg of FUT8 or GFT siRNA expression vector linearized at the FspI site (FT8lib3/pAGE or GFT_3G10/pAGE). Transfectants were selected in 3 μg/mL puromycin and 400 μg/mL hygromycin (Wako Pure Chemical Industries, Osaka, Japan) for 8 days, and then the drug-resistant clones were subjected to 7-day selection with 500 μg/mL LCA in the presence of 100 μM L-fucose. The resultant LCA-resistant clones were isolated and expanded in IMDM medium containing 10% (v/v) dFBS, 500 nM MTX, 3 μg/mL puromycin, and 400 μg/mL hygromycin for further real-time PCR and antibody analyses, as described above.

The CHO/DG44 32-05-12 (1.6 × 10⁶) cells were transformed by electroporation with 10 μg of the FUT8 and GMD siRNA tandem expression vector linearized at the FspI site (FT8lib3_GMDB/pAGE). Transfectants were selected in 500 μg/mL hygromycin for 8 days, and then the drug-resistant clones were subjected to 7-day selection with 500 μg/mL LCA and 1 mM L-fucose. The resultant LCA-resistant clones were isolated and expanded in IMDM medium containing 10% (v/v) dFBS, 500 nM MTX, and 500 μg/mL hygromycin for further real-time PCR and antibody analyses as described above. The established clones were adapted to serum-free medium EX-CELL™ 302 (JRH Biosciences) supplemented with 6 mM L-glutamine, 500 nM MTX, and 500 μg/mL hygromycin for LCA-reactivity analysis and serum-free fed-batch culture.

A 1L-scale spinner bioreactor (ABLE, Tokyo, Japan) was employed to maintain controlled, serum-free, fed-batch culture conditions of 35°C, pH 7.1, 50% DO. Each clone was inoculated at 3.0 × 10⁵ cells/mL in EX-CELL™302 medium supplemented with 6 mM L-glutamine and 500 nM MTX, and was fed with serum-free IMDM-based feeding medium containing 5.3 μM human recombinant insulin (Invitrogen) at days 3, 5, 7, 9, and 11 post-inoculation to maintain a glucose content of approximately 5.0 g/L. Culture aliquots were drawn on days 3, 6, 9, 12, 14, and 16 prior to addition of the feeding medium. Viable cell density was measured with an automatic cell counter, Vi-CELL™ XR (Beckman Coulter, Fullerton, CA), using trypan blue exclusion. The antibody concentration in the culture supernatant was measured by an enzyme-linked immunosorbent assay (ELISA) specific for human IgG1, as previously described 38.