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IPT 25 2008 24/4/08 09:44 Page 50 Biopharma Human Cell-Expressed Proteins The production of human recombinant proteins from human cells provides some unique advantages for clinical applications; these are imparted by human-specific glycosylation patterns and include lower immunogenicity, greater biological activity and greater stability both in vitro and in vivo By Denese Marks and Glenn Pilkington at Apollo Life Sciences Dr Denese Marks received a PhD in Biochemistry from the University of Technology, Sydney (Australia), and has subsequently carried out postdoctoral research at the University of Vienna (Austria), the University of Melbourne (Australia) and the Institute of Child Health (London, UK), before joining Apollo Cytokine Research as Head of Cell Biology in 2005. Glenn Pilkington has spent fourteen years in the biotechnology industry at Diadexus, LLC (South San Francisco, USA), Intracel Corporation (Frederick, USA), and two years at Apollo Cytokine Research (Sydney, Australia) in the development of fully human therapeutic proteins. He obtained his MSc from the University of Melbourne (Australia) and is a co-inventor on 22 patent applications, including 12 relating to Apollo’s hcxTM technology. The use of human proteins as biopharmaceuticals in the treatment of disease – including stem cell transplantation and immunotherapy for cancer – has increased dramatically over the past three decades. During the same period, regulatory bodies such as the FDA and EMEA have indicated a preference for nonanimal derived supplements, recommending that the industry use well-characterised cell lines for the production of human protein biopharmaceuticals. The production of proteins in human cells also provides some unique advantages for clinical applications, including greater activity and stability in vitro and in vivo, and lower immunogenicity imparted by the human-specific glycosylation. These benefits are often not available from the non-human expression systems currently used to produce therapeutic proteins, such as bacteria, yeast, insect, mouse or hamster cells. The treatment of several diseases, particularly cancer, now routinely includes cell-based therapies whereby human cells are harvested, cultured ex vivo and re-infused into patients. Ex vivo expansion of human cells requires chemically defined media containing optimal combinations of recombinant human cytokines and growth factors. Most growth factors and cytokines are highly glycosylated, with glycosylation representing up to 75% of their molecular weight. Glycosylation is a highly species-specific post-translational modification that affects the biological properties of proteins. Recombinant proteins produced from E. coli are not glycosylated, and growth factors and cytokines produced from other expression systems – such as insect, yeast or mammalian cells – exhibit different glycosylation patterns compared with native human proteins. The importance of humanspecific glycosylation of cytokines and growth factors is now being recognised, as differences in glycosylation can 50 cause the alteration of a protein’s biological properties, resulting in higher immunogenicity, lower biological activity and altered pharmacodynamics in vivo. With the trend to elimination of non-human components from biopharmaceutical production, human cell-expressed proteins offer unique advantages. At Apollo Cytokine Research, we produce human cell-expressed (hcxTM) proteins with human-specific post-translational modifications that are as close as possible to the native protein. Our aim is to identify structural and biological differences between these proteins and equivalent proteins produced from non-human expression systems, and we routinely evaluate differences in cell proliferation, viability, morphology, cell surface marker expression, and activation of specific intracellular signalling pathways. This article provides an overview of how hcxTM proteins can provide benefits for human cell culture, focusing in particular on EPO (erythropoietin), G-CSF granulocytecolony stimulating factor, SCF (stem cell factor) and IL-4 (interleukin-4) for culturing haematopoietic stem cells (HSCs) and dendritic cells (DCs). CULTURE OF HUMAN HAEMATOPOIETIC STEM CELLS All mature blood and immune cells develop from a population of adult stem cells called haematopoietic stem cells (HSCs). They are generally characterised by expression of the CD34 cell surface antigen; these CD34 positive (CD34+) cells have the capacity for self-renewal and differentiation into a variety of specialised cell types. The regenerative capacity of these cells is finely controlled by the cellular micro-environment, and through molecular signals from cytokines and growth factors. Ex vivo expanded HSCs are used therapeutically for stem cell transplantation following myelo-ablative Innovations in Pharmaceutical Technology 24/4/08 09:44 Page 51 chemotherapy or radiotherapy, as well as for the treatment of blood disorders such as anaemia and thalassaemia. It is necessary to expand HSCs ex vivo to generate sufficient cells to be of therapeutic benefit. Some important benefits of culturing CD34+ HSCs in TM EPOhcx, G-CSFhcx or SCFhcx are outlined below. G-CSF and SCF Granulocyte colony stimulating factor (G-CSF) stimulates differentiation and maturation of granulocytic cells, and activates neutrophils in the circulation. G-CSF has one O-linked glycosylation site, which is known to increase the stability of G-CSF (1), resulting in higher biological activity and greater ability to stimulate CD34+ stem cell proliferation (2). G-CSF is used in combination with other cytokines and growth factors for ex vivo expansion of CD34+ stem cells to treat post-chemotherapy neutropenia in cancer patients (3,4). G-CSF is also used to mobilise stem cells from cancer patients or normal donors for stem cell transplantation and reconstitution (5). Mobilisation with glycosylated G-CSF results in neutrophils with normal morphology and function, whereas mobilisation with nonglycosylated G-CSF has resulted in neutrophils with defective chemotaxis and increased actin polymerisation (6,7), emphasising the importance of glycosylation on therapeutic proteins. Stem cell factor (SCF) is another glycoprotein that augments proliferation of erythroid, myeloid and lymphoid haematopoietic progenitors ex vivo. SCF acts synergistically with other cytokines and is known to protect progenitor cells from apoptosis (8) and to prime stem cells for differentiation. SCF is heavily glycosylated, with N- and O-linked glycans on multiple residues. SCF is routinely used clinically in combination with other cytokines to expand stem cells ex vivo, and in combination with G-CSF to mobilise CD34+ cells into the circulation of cancer patients (4). To examine the benefits of glycosylation, the ability of glycosylated G-CSFhcx was tested in combination with SCFhcx for expansion of CD34+ human HSCs in vitro and compared with commercially available non-glycosylated recombinant growth factors produced in E. coli. The glycosylated G-CSFhcx and SCFhcx induced enhanced proliferation, producing more and larger colonies in colony-forming assays, compared with G-CSF and SCF from E. coli (see Figure 1). When used in combination in liquid culture, the glycosylated G-CSFhcx and SCFhcx promoted significantly more cellular proliferation than E. coli-expressed G-CSF and SCF. Preliminary evidence suggests that G-CSFhcx was a more potent activator of STAT3, which may be the underlying cell signalling mechanism responsible for the increased cell Innovations in Pharmaceutical Technology 70 60 Number of colonies IPT 25 2008 50 40 30 20 10 0 E coli Small hcx Large Total Colony size proliferation. G-CSFhcx and SCFhcx, when used together, form a more potent cocktail for HSC expansion in vitro. The increased biological activity of these growth factors could translate into more effective clinical therapies. For example, the enhanced ex vivo amplification of CD34+ cells using hcxTM growth factors could reduce the period of neutropenia following stem cell transplantation, subsequently reducing the incidence of infection and increasing the survival rates of patients. Erythropoietin Erythropoietin (EPO) is one of the most studied molecules in terms of the requirement for glycosylation for correct protein function. It is used predominantly to treat anaemia resulting from kidney disease or chemotherapy/radiotherapy as it induces erythroid differentiation of CD34+ cells, leading to the generation of mature red blood cells in vivo. EPO is heavily glycosylated with one O-linked and 3 N-linked glycosylation sites, and an average oligosaccharide content of 40% by weight. Glycosylation of EPO is important for cellular secretion, and protein conformation, solubility, stability and biological activity (9). In particular, terminal sialylation on the N-glycans of EPO is necessary for increased circulatory half-life, as asialo-erythropoietin is rapidly cleared from the plasma (10). Until recently, most commercially available recombinant human EPO was purified from Chinese Hamster Ovary (CHO) or Baby Hamster Kidney (BHK) cells. However, some humanspecific oligosaccharide structures are not synthesised by CHO or BHK cells, as they lack several glycosyl transferases including sialyl-α 2-6 transferase and α 1-3/4 fucosyl transferase (11). The absence of these enzymes in non-human cell lines may be detrimental to the activity of EPO. These potential differences thus make structural analysis of glycans and their subsequent correlation with biological activity of great importance. Our research indicated that EPOhcx, in combination with stem cell factor (SCFhcx), induced over two-fold more erythroid differentiation of CD34+ HSCs, compared with Figure 1: Clonogenic activity of CD34+ expanded cells by G-CSF and SCF. Expanded CD34+ HSCs were inoculated into semisolid cultures of methylcellulose containing G-CSFhcx and SCFhcx or E. coli-expressed G-CSF and SCF. Cultures were incubated at 37ºC and the number and sizes of colonies scored 14 days later using an inverted microscope 51 IPT 25 2008 24/4/08 09:45 Page 52 90 a) Glycophorin A expression 250 EPOhcx CHO EPO 200 Events Mean fluorescence units 300 150 100 50 0 0 100 101 102 103 104 Fluorescence intensity 1,800 EPOhcx 1,600 CHO EPO 1,400 1,200 1,000 800 Events Mean fluorescence units 2,000 90 b) CD71 expression 600 400 for therapeutic applications. Hence DCs must be expanded ex vivo from precursor cells, such as peripheral blood monocytes, in order to generate vaccines. Production of ‘clinical grade’ DCs for large-scale cancer vaccine development follows current good manufacturing practice (cGMP) guidelines. These protocols use serumfree medium to eliminate non-human proteins, but continue to use cytokines and growth factors such as GM-CSF, IL-4 and TNF-α produced from non-human cells. The use of hcxTM proteins in these processes would eliminate all animal-derived products and provide other benefits, such as greater stability in cell culture. Glycosylated IL-4hcx is more stable in cell culture medium than the non-glycosylated IL-4 currently used. Increased stability in culture has the advantage of reducing cost and the number of medium changes, thereby reducing the risk of contamination. 0 0 200 100 101 102 103 104 Fluorescence intensity Figure 2: EPO-induced erythroid differentiation. CD34+ HSCs were cultured with EPOhcx and SCFhcx or CHO cellexpressed EPO and SCF for 7 days, and the expression of erythroid markers (a) glycophorin A and (b) CD71 was determined CHO-expressed EPO. This was demonstrated by higher expression of both glycophorin A (see Figure 2a) and CD71 (see Figure 2b), which are markers of more mature erythroid differentiation. We also observed that CD34+ HSCs cultured in EPOhcx produced morphologically more differentiated cells than CD34+ HSCs grown in CHO-expressed EPO, which remained blast-like. Similarly, haemoglobin expression was higher in HSCs grown in EPOhcx, compared with CHO-expressed EPO. Cellular proliferation and viability was, however, equivalent with both sources of EPO – indicating that the increased cellular differentiation using EPOhcx did not compromise cell number or viability in these cultures. Thus, our in vitro results using EPOhcx demonstrated more rapid induction of erythroid differentiation of HSCs compared with non-human cell-expressed EPO, again suggesting potential clinical benefits such as the production of more functionally mature erythroid cells in EPOhcx-treated patients. DENDRITIC CELL CULTURE Dendritic cells (DCs) are potent antigen-presenting cells of the immune system and are actively involved in immune responses, including graft rejection and T-cell dependent responses, as well as in autoimmune diseases and the pathogenesis of HIV infection (12). DCs are used clinically to generate vaccines against several cancers, whereby autologous DCs are generated ex vivo and then pulsed with tumour-related peptides, proteins or tumour cell lysates to boost antigen-specific T-cell mediated tumour immunity. DCs represent a small percentage of leukocytes and cannot be isolated in sufficient numbers 52 CONCLUSION Human cell-expressed proteins offer the benefits of fully human proteins with human-specific post-translational modifications, reducing the potential for immunogenicity. Other benefits include elimination of the risk of transmissible spongiform encephalopathies (TSE) and other infectious diseases, a longer half-life in vitro and in vivo, and often greater biological activity. The growing requirement for chemically defined, animal protein-free media in the production of biopharmaceuticals suggests that human cell-expressed proteins will provide the next generation of growth factors and cytokines for ex vivo and in vivo therapeutic applications. The authors can be contacted at [email protected] References 1. Carter et al, Biologicals. 32, pp37-47, 2004 2. Qerol et al, Hematologica. 84, pp493-498, 1999 3. Reiffers et al, Lancet. 354, pp1,092-1,093, 1999 4. McNeice et al, Blood. 96, pp3,001-3,007, 2000 5. Anderlini and Champlin, Drugs. 62 Suppl. 1, pp79-88, 2002 6. Azzara et al, Am. J. Hematol. 66, pp306-307, 2001 7. Carulli et al, Am. J. Hematol. 81, pp318-323, 2006 8. Zeuner et al, Blood. 102, pp87-93, 2003 9. Egrie and Brown, 2001 10. Fukuda et al, Blood. 73, pp84-89, 1989 11. Skibeli et al, Blood. 98, pp3,626-3,634, 2001 12. Steinman, Annu. Rev. Immunology. 9, pp274-296, 1991 13. Casadevall, Nephrol. Dial. Transplant. 16 (suppl. 3), pp3-13, 2005 Innovations in Pharmaceutical Technology