Evaluation of expression systems of recombinant human interferon gamma

Razaghi, Ali (2017) Evaluation of expression systems of recombinant human interferon gamma. PhD thesis, James Cook University.

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Human interferon gamma (hIFNγ) is a cytokine belonging to a diverse group of interferons which have a crucial immunological function against mycobacteria and a wide variety of viral infections. Specifically, recombinant hIFNγ has been shown to be an effective biopharmaceutical, against a wide range of viral, immuno-suppressive diseases with promising prospects in cancer immunotherapy resulting in a strong increase in demand and price. To date, it has been approved for treatment of chronic granulomatous disease and malignant osteopetrosis. hIFNγ is commonly expressed in Escherichia coli, marketed as ACTIMMUNE®. However, the resulting product of the prokaryotic expression system is unglycosylated with a short half-life in the bloodstream; the purification process is tedious and makes the product costlier. To solve these limitations; recombinant hIFNγ, as a lucrative biopharmaceutical, has been engineered in different expression systems including prokaryotic, protozoan, fungal (yeasts), plant, insect, and mammalian cells. Other expression systems also did not show satisfactory results regarding yields, the biological activity of the protein or economic viability. This thesis aimed to 1) lower the cost of production by using cheap C1 carbon sources (e.g. methane) from agricultural activities (e.g. intensive dairy, piggeries, etc.) for the cultivation of transformed yeast and 2) to assess the therapeutic efficacy of recombinant hIFNγ in its glycosylated and non-glycosylated form from different expression systems against ovarian cancer cells. Chapter 1 of the thesis gives a comprehensive review of expression and production of recombinant hIFNγ leading to the aims of the research. The second chapter investigated the potential of Rhodotorula glutinis; a yeast once reported as a methane-oxidizing yeast, for growth on cheap C1 carbon sources (methane and methanol) to evaluate the species potential for lowering production costs of recombinant immuno-therapeutics. In contrast to previous reports, R. glutinis did not utilise any C1 carbon sources even under near-identical experimental conditions to those reported. It also failed to grow on intermediates of the methane oxidation pathway (methanol, formaldehyde and formate) and only grew on C2 or more complex carbon sources. It is therefore concluded that R. glutinis is neither a methanotrophic nor methylotrophic yeast and not suitable as a cheap carbon-sustained expression system. This result led the research to look for an alternative yeast species with a proven ability to utilise a C1 carbon source (i.e. methanol). Among these alternative expression systems, Pichia pastoris was chosen as a proven methylotrophic (i.e. methanol-utilising) heterologous expression system. Six months after choosing this expression system, efficient expression of hIFNγ was reported by Wang et al (2014). Therefore, the third chapter replicated hIFNγ expression in P. pastoris similar to the previous study and expanded on it by using four different strains (X33: wild type; GS115: HIS-Mut+; KM71H: Arg+, Mut- and CBS7435: Mutˢ) and three different vectors (pPICZαA, pPIC9, and pPpT4αS). In addition, the native sequence (NS) and two codon-optimised sequences (COS1 and COS2) for P. pastoris were used. Methanol induction yielded no expression/ secretion of hIFNγ in X33; highest levels were recorded for CBS7435: Mutˢ (~16 μg L⁻¹). mRNA copy number calculations acquired from RT-qPCR for GS115-pPIC9-COS1 proved low abundance of mRNA. A 10-fold increase in expression of hIFNγ was achieved by lowering the minimal free energy of the mRNA and a 100-fold by using the Mutˢ phenotypes, but yields were substantially lower than reported by Wang et al (2014). The results show that commercial production of low cost, eukaryotic recombinant hIFNγ is not an economically viable in P. pastoris. In the fouth chapter, the aim was to study how selective pressure on the Histidinol dehydrogenase gene (HIS4), using amino acid starvation, affects the level of expression and secretion of the adjacent hIFNγ in the transformed P. pastoris GS115 strain, a histidine-deficient mutant. hIFNγ was cloned into the pPIC9 vector adjacent to the HIS4 gene, a gene essential for histidine biosynthesis, which was then transformed into P. pastoris. Under amino acid starvation, only successfully transformed cells (hIFNγ –HIS4+) can synthesise histidine and therefore thrive. As shown by ELISA, amino acid starvation-induced selective pressure on HIS4 improved expression and secretion of the adjacent hIFNγ by 55% compared to unchallenged cells. RT-qPCR showed that there was also a positive correlation between duration of amino acid starvation and increased levels of the hIFNγ RNA transcripts. According to these results, it is suggested that these adjacent genes (hIFNγ and HIS4) in the transformed P. pastoris are transcriptionally co-regulated and their expression is synchronised. To the best of the knowledge of the authors; this is the first study demonstrating that amino acid starvation-induced selective pressure on HIS4 can alter the regulation pattern of adjacent genes in HIS4⁺ P. pastoris strains. The aim of the fifth chapter was to determine the effect of glycosylation and expression platform of hIFNγ on ovarian carcinoma cell lines; PEO1 & SKOV3. Additionally, signalling transduction pathway for cytostasis and cell death were explored. The results showed that hIFNγ affected both PEO1 and SKOV3, but the E. coli-derived product was not effective against SKOV3, while the mammalian expressed was effective against both cancer cell lines. The primary effect was through cytostasis by cell cycle arrest and to a lesser extent through cytotoxicity, whilst the cell death mechanism was not apoptotic. Mammalian expressed hIFNγ, particularly when expressed in HEK293 (human embryonic kidney 293), showed better cytostatic effectiveness for both cell lines and higher cytotoxicity towards SKOV3. Furthermore, deglycosylation only slightly reduced the cytostatic and cytotoxic effects of the CHO-expressed hIFNγ. In general; mammalian expressed hIFNγ may be advantageous for inhibiting the growth of ovarian carcinomas more effectively, particularly for drug-resistant cell lines. We also suggested for the first time that upregulation of FADD in SKOV3 can be the reason of anti-apoptotic behaviour and drug resistance in this cell line, which may present a novel therapeutic target. In conclusion, expression of hIFNγ in C1 carbon utilising yeast yielded insufficient product to be commercially viable. I, therefore, recommend exploring different mammalian expression systems e.g. CHO, HEK293, PER.C6, and CAP/CAP-T for the production of this biopharmaceutical because these expression systems are highly productive, cost-efficient, possess human-like post-translation glycosylation outcomes which increase biological activity and half-life of the protein in the bloodstream. Achieving the milestone of improved quality and lowered costs can also facilitate uptake of mammalian-expressed recombinant hIFNγ for clinical trials particularly due to a strong potential in cancer immunotherapy.

Item ID: 51928
Item Type: Thesis (PhD)
Keywords: actimmune, biopharmaceuticals, cancer immunotherapy, Gcn4p, glycosylation, histidinol dehydrogenase, human interferon gamma, interferon gamma, low-abundance RNA, minimum free energy, ovarian cancer, Pichia pastoris, protein expression and purification, serial passage, transcriptional co-regulation
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Publications arising from this thesis are available from the Related URLs field. The publications are:

Chapter 1: Razaghi, Ali, Owens, Leigh, and Heimann, Kirsten (2016) Review of the recombinant human interferon gamma as an immunotherapeutic: Impacts of production platforms and glycosylation. Journal of Biotechnology, 240. pp. 48-60.

Chapter 3: Razaghi, Ali, Tan, Emylin, Lua, Linda H.L., Owens, Leigh, Karhikeyan, O.P., and Heimann, Kirsten (2017) Is Pichia pastoris a realistic platform for industrial production of recombinant human interferon gamma? Biologicals, 45. pp. 52-60.

Chapter 4: Razaghi, Ali, Huerlimann, Roger, Owens, Leigh, and Heimann, Kirsten (2015) Increased expression and secretion of recombinant hINFγ through amino acid starvation-induced selective pressure on the adjacent HIS4 gene in Pichia pastoris. Acta Facultatis Pharmaceuticae Universitatis Comenianae, 62 (2). pp. 1-8.

Date Deposited: 09 Jan 2018 22:21
FoR Codes: 11 MEDICAL AND HEALTH SCIENCES > 1112 Oncology and Carcinogenesis > 111201 Cancer Cell Biology @ 25%
11 MEDICAL AND HEALTH SCIENCES > 1112 Oncology and Carcinogenesis > 111207 Molecular Targets @ 25%
06 BIOLOGICAL SCIENCES > 0604 Genetics > 060412 Quantitative Genetics (incl Disease and Trait Mapping Genetics) @ 50%
SEO Codes: 86 MANUFACTURING > 8608 Human Pharmaceutical Products > 860803 Human Pharmaceutical Treatments (e.g. Antibiotics) @ 100%
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