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Engineering CELLS,
   Engineering RESPONSES

EXPRESSION ENGINEERING

Efficacy & Safety

Glycosylation is the most common post-translational modification of proteins. Many recombinant protein drugs require proper glycosylation in order to be fully functional. The glycans attached to the biotherapeutics can have a dramatic impact on the efficacy and safety of glycoprotein drugs. Proteins such as EPO require high levels of sialylation on their N-glycans as the lack of sialylation results in their rapid removal from the circulation by liver cells.

Alternatively, the lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human Fc receptor and consequently enhances antibody-dependent cellular toxicity (ADCC). Recombinant glucocerebrosidase (Cerezyme) can enter its target cells, macrophages, only if it carries mannose-terminated N-glycans. These observations highlighted the need for engineering the glycosylation pathways in the host cells in order to produce recombinant protein drugs with optimal and consistent glycans. In Expression Engineering Group 1 (EE-1), our goal is to improve the efficacy and safety of recombinant therapeutics by manipulating the glycosylation pathways in mammalian cells.

Specifically, we aim to generate host cells for the production of therapeutic biologics with enhanced efficacy. Using cytotoxic lectins and zinc-finger nuclease (ZFN) technologies (see figure), our group has isolated/generated novel CHO glycosylation mutants that can produce fucose-free antibodies, recombinant proteins with highly sialylated N-glycans and proteins with mannose-terminated N-glycans.

The number of recombinant monoclonal antibodies approved for the treatment of cancer and inflammatory diseases has dramatically increased in recent years. The biotech industry faces a major challenge in meeting this demand due to the inability to produce recombinant antibodies at high levels. To address these problems, we have developed a technology to optimize the signal peptides for 5 best-selling antibody drugs to permit their efficient secretion and/or production in CHO cells. In collaboration with other groups in BTI, we are currently developing technologies to produce these antibodies as biosimilar therapeutics.

Our group also directs efforts towards investigating the molecular mechanisms that underlies the function, regulation and localization of glycosyltransferases and nucleotide sugar transporters in mammalian cells. Novel techniques have been developed to study the structure-functional relationships of these proteins.

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Gene Expression and Beyond

The Expression Engineering II team, which is one of the upstream R&D groups in BTI, adopts various molecular, cellular, and biochemical approaches to investigate the regulatory mechanisms of transcription and cell cycle progression. With intense internal and external research collaborations, the team focuses on identifying targets for potential development of innovative anti-cancer strategies and refining tactics in improving the yield of recombinant biopharmaceutical proteins.

The team is currently investigating on hexamethylene bisacetamide inducible protein 1 (HEXIM1). HEXIM1 is best-known as the inhibitor of positive transcription elongation factor b (P-TEFb), which controls transcription elongation of RNA polymerase II. In recent years, the team has unraveled that HEXIM1's role is not only limited to transcriptional regulation, but also prevailed in cell cycle regulation. HEXIM1 is identified as a positive regulator of p53, found to associate with two other p53 regulators (HDM2 and nucleophosmin), involved in tumorigenesis of acute myeloid leukemia (through interaction with the cytoplasmic NPM mutant, NPMc+), and participated in the differentiation of human pluripotent stem cells.

Another key interest of the team is the regulation of X-box binding protein 1S (XBP-1S). XBP-1S is a spliced active form of XBP-1 and an essential factor of unfolded protein response. XBP-1S has been shown to increase recombinant protein production and activate the transcription of an oncovirus, human T-lymphotropic virus 1. The group is currently exploring the potential of a novel anti-cancer approach through post-translational modification of XBP-1S.

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People

Dr. Song Zhiwei

PhD in Biochemistry (1993) University of Michigan, Ann Arbor, USA

Research Focus/Interests
  • Glycosylation of recombinant protein drugs
  • Apoptosis in cultured mammalian cells
  • Developing cell lines to produce follow-on biologics such as therapeutic antibodies

Dr. Jimmy Chao Sheng-Hao

PhD in Molecular Biology (2001) University of Iowa, USA

Research Focus/Interests
  • Regulation of cellular and viral transcription
  • Potential use of a novel HEXIM1 cytotoxic peptide in anti-cancer and anti-microbial therapy
  • Role of unfolded protein response in viral replication
  • Recombinant protein and peptide production

Lin Pao Chun

Dr. Lin Pao Chun

PhD in Molecular Cell Biology (2011), NUS Graduate School of Integrative Sciences and Engineering, National University of Singapore, Singapore

Research Focus/Interests
  • Developing cell lines for the production of therapeutic proteins
  • Evaluating the mechanisms of glycosylation and its impact on diseases

Dr. Wang Huashan

PhD in Molecular genetics (2006), Yong Loo Lin School of Medicine, National University of Singapore, Singapore

Research Focus/Interests
  • Expression of anticancer antibodies in cell lines
  • Development of cell lines for the production of therapeutic antibodies

Dr. Irene Kiess

PhD in Cancer Biology (2010), Karlsruhe Institute of Technology, Germany

Research Focus/Interests
  • Glycosylation of recombinant therapeutic proteins and its role in stability, solubility and biological activity
  • Generation of highly expressing stable cell lines for the production of next-generation biologics

Dr. Tan Yun Lei

PhD in Chemical Biology (2014), The Scripps Research Institute, La Jolla, USA

Research Focus/Interests
  • Development of cell lines to produce glycoprotein biologics with enhanced efficacy and safety

Dr. Wong Sze Yue

PhD in Bioengineering (2016), University of California Berkeley, USA

Research Focus/Interests
  • Glycosylation of therapeutic proteins produced in mammalian cells for improved safety and efficacy
  • High expressing stable cell line generation

Year 2018

    Natasha A. Pereira, Kah Fai Chan, Pao Chun Lin and Zhiwei Song (2018) The “less-is-more” in therapeutic antibodies: Afucosylated anti-cancer mAbs for enhanced ADCC. mAbs 10(5): 693-711

    Natasha A. Pereira, Kah Fai Chan, Pao Chun Lin and Zhiwei Song (2018) The “less-is-more” in therapeutic antibodies: Afucosylated anti-cancer mAbs for enhanced ADCC. mAbs 10(5): 693-711

    Kian Ping Chan, Sheng-Hao Chao, and James Chen Yong Kah (2018) Universal mRNA Translation Enhancement with Gold Nanoparticles Conjugated to Oligonucleotides with a Poly(T) Sequence. ACS Applied Materials & Interfaces 10(6): 5203-5212

Year 2017

    Han Kee Ong, Benjamin P.C. Soo, Kai Ling Chu, and Sheng-Hao Chao (2017) XBP-1, a cellular target for the development of novel anti-viral strategies. Current Protein & Peptide Science (Published Online)

    Jian'er Lin, Shu Hui Neo, Steven C. L. Ho, Jessna H. M. Yeo, Tianhua Wang, Wei Zhang, Xuezhi Bi, Sheng-Hao Chao and Yuansheng Yang (2017) Impact of Signal Peptides on Furin-2A Mediated Monoclonal Antibody Secretion in CHO Cells. Biotechnology Journal 12: 9

    Benjamin P.C. Soo, Julian Tay, Shirelle Ng, Steven C.L. Ho, Yuansheng Yang, and Sheng-Hao Chao (2017) Correlation between expression of recombinant proteins and abundance of H3K4Me3 on the enhancer of human cytomegalovirus major immediate-early promoter. Molecular Biotechnology 59(8): 315-322

    Kian Ping Chan, Yang Gao, Jeremy Xianwei Goh, Dewi Susanti, Eugenia Li Ling Yeo, Sheng-Hao Chao and James Chen Yong Kah (2017) Exploiting the Protein Corona on DNA Functionalized Gold Nanoparticles for Enhanced mRNA Translation. ACS Applied Materials & Interfaces 9(12): 10408-10417

    Jinjia Wang, Xiaolong Wang, Lei Shi, Fei Qi, Ping Zhang, Yuanxing Zhang, Xiangshan Zhou, Zhiwei Song & Menghao Cai (2017) Methanol-Independent Protein Expression by AOX1 Promoter with trans-Acting Elements Engineering and Glucose-Glycerol-Shift Induction in Pichia pastoris. Scientific Reports 7: 41850

Year 2016

    Steven C. L. Ho, Esther Y. C. Koh, Benjamin P. C. Soo, Mariati, Sheng-Hao Chao and Yuansheng Yang (2016) Evaluating the use of a CpG free promoter for long-term recombinant protein expression stability in Chinese hamster ovary cells. BMC Biotechnology 16: 71

    Peiqing Zhang, Susanto Woen, Tianhua Wang, Brian Liau, Sophie Zhao, Chen Chen, Yuansheng Yang, Zhiwei Song, Mark R. Wormald, Chuanfei Yu and Pauline M. Rudd (2016) Challenges of glycosylation analysis and control: an integrated approach to producing optimal and consistent therapeutic drugs. Drug Discovery Today 21(5): 740-765

    Shu Hui Neo, Qiao Jing Lew, Ser Mei Koh, Lu Zheng, Xuezhi Bi, and Sheng-Hao Chao (2016) Use of a novel cytotoxic HEXIM1 peptide in the directed breast cancer therapy. Oncotargets 7(5): 5483-5494

Year 2015

    Kah Fai Chan, Wahyu Shahreel, Corrine Wan, Gavin Teo, Noor Hayati, Shi Jie Tay, Wen Han Tong, Yuansheng Yang, Pauline M. Rudd, Peiqing Zhang and Zhiwei Song (2015) Inactivation of GDP-fucose transporter gene (Slc35c1) in CHO cells by ZFNs, TALENs and CRISPR-Cas9 for production of fucose-free antibodies. Biotechnology Journal 11(3): 399-414

    Steven C. L. Ho, Tianhua Wang, Zhiwei Song and Yuansheng Yang (2015) IgG aggregation mechanism for CHO cell lines expressing excess heavy chains and strategies to reduce the aggregates. Molecular Biotechnology 57(7): 625-634

    Ning Li, Laura Sutarlie, Qiao Jing Lew, Sheng-Hao Chao and Xiaodi Su (2015) Identification of Lysine K18 Acetylation on Histone H3 Peptide Using Gold Nanoparticles’ Aggregation Behaviour. Amino Acids (Published Online)

    Ryan Haryadi, Steven Ho, Yee Jiun Kok, Helen X. Pu, Lu Zheng, Natasha A. Pereira, Bin Li, Xuezhi Bi, Lin-Tang Goh, Yuansheng Yang, Zhiwei Song (2015) Optimization of Heavy Chain and Light Chain Signal Peptides for High Level Expression of Therapeutic Antibodies in CHO Cells. Public Library of Science (PLOS) 10(2): e0116878

Year 2014

    John SY Goh, Yingwei Liu, Kah Fai Chan, Corinne Wan, Gavin Teo, Peiqing Zhang, Yuanxing Zhang and Zhiwei Song (2014) Producing recombinant therapeutic glycoproteins with enhanced sialylation using CHO-gmt4 glycosylation mutant cells. Bioengineered 5(4): 269–273

    Qiao Jing Lew, Kai Ling Chu, Yi Ling Chia, Benjamin Soo, Jia Pei Ho, Chew Har Ng, Hui Si Kwok, Cheng-Ming Chiang, Yao Chang and Sheng-Hao Chao (2014) GCN5 inhibits XBP-1S-mediated transcription by antagonizing PCAF action. Oncotarget 6(1): 271-287

    Kah Fai Chan, John SY Goh and Zhiwei Song (2014) Improving sialylation of recombinant biologics for enhanced therapeutic efficacy. Pharmaceutical Bioprocessing 2(5): 363–366 (Editorial)

    Beng Hui Tan, Yasutsugu Suzuki, Hirotaka Takahashi, Ho Rui Ying Pamela, Chikako Takahashi, Qi’En Han, Wei Xin Chin, Sheng-Hao Chao, Tatsuya Sawasaki, Naoki Yamamoto, and Youichi Suzuki (2014) Identification of RFPL3 as a novel E3 ubiquitin ligase modulating the integration activity of human immunodeficiency virus type 1 preintegration complex using a newly developed protein screening system. Journal of Biological Chemistry 289: 26368-26382

    Susana Soo-Yeon Kim, Koon-Guan Lee, Ching-Siang Chin, Say-Kong Ng, Natasha Ann Pereira, Shengli Xu, and Kong-Peng Lam (2014) DOK3 is required for IFN-β production by enabling TRAF3/TBK1 complex formation and IRF3 activation. Journal of Immunology 193(2): 840-848

    Yingwei Liu, Xiangshan Zhou, Zhiwei Song, and Yuanxing Zhang (2014) Sodium butyrate enhances the acidic isoform content of recombinant human erythropoietin produced by Chinese hamster ovary cells. Biotechnology Letters 36(5): 907-911

    Natasha A. Pereira, Helen X. Pu, Hazel Goh and Zhiwei Song (2014) Golgi phosphoprotein 3 mediates the Golgi localization and function of protein O-linked mannose β-1,2-Nacetlyglucosaminyltransferase 1.The Journal of Biological Chemistry 289: 14762-14770

    Ruichuan Chen, Jasper H. N. Yik, Qiao Jing Lew, and Sheng-Hao Chao (2014) Brd4 and HEXIM1: Multiple roles in P-TEFb regulation and cancer. BioMed Research International 232870: 1-11

    Yi Ling Chia, Chew Har Ng, Philip Lashmit, Kai Ling Chu, Qiao Jing Lew, Jia Pei Ho, Hsueh Lee Lim, Peter Morin Nissom, Mark F. Stinski and Sheng-Hao Chao (2014) Inhibition of human cytomegalovirus replication by overexpression of CREB1. Antiviral Research 102: 11–22

Year 2013

    Kai Ling Chu, Qiao Jing Lew, Vikneswari Rajasegaran, Jing Ting Kung, Lu Zheng, Qiaoyun Yang, Rachel Shaw, Nge Cheong, Yih-Cherng Liou and Sheng-Hao Chao (2013) Regulation of PRDX1 peroxidase activity by Pin1. Cell Cycle 12(6):944–952

    Esther Y. C. Koh, Steven C. L. Ho, Mariati, Zhiwei Song, Xuezhi Bi, Muriel Bardor and Yuansheng Yang (2013) An internal ribosome entry site (IRES) mutant library for tuning expression level of multiple genes in mammalian cells. PLOS One 8(12): e82100

    Qiao Jing Lew, Kai Ling Chu, Yi Ling Chia, Nge Cheong and Sheng-Hao Chao (2013) HEXIM1, a New Player in the p53 Pathway. Cancers 5(3): 838-856

    Vanessa Ding, Qiao Jing Lew, Kai Ling Chu, Subaashini Natarajan, Vikneswari Rajasegaran, Meera Gurumurthy, Andre B. H. Choo and Sheng-Hao Chao (2013) HEXIM1 induces differentiation of human pluripotent stem cells. PlosOne 8(8): e72823

    John S.Y. Goh, Yingwei Liu, Haifeng Liu, Kah Fai Chan, Corrine Wan, Gavin Teo, Xiangshan Zhou, Fusheng Xie, Peiqing Zhang, Yuanxing Zhang and Zhiwei Song (2013) Highly sialylated recombinant human erythropoietin production in large-scale perfusion bioreactor utilizing CHO-gmt4 (JW152) with restored GnT I function. Biotechnology Journal 9: 100–109

    Steven C. L. Ho, Muriel Bardor, Bin Li, Jia Juan Lee, Zhiwei Song, Yen Wah Tong, Lin-Tang Goh, Yuansheng Yang (2013) Comparison of internal ribosome entry site (IRES) and furin-2A (F2A) for monoclonal antibody expression level and quality in CHO cells. PlosOne 8(5): e63247

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