Interleukin-2 mutein expressed from mammalian cells

[0002] Related subject matter is disclosed in patent application Ser. No. 09/080,080, entitled “IL-2 Selective Agonists and Antagonists,” filed May 15, 1998 in the names of Shanafelt et al., the entirety of which is incorporated herein by reference.

[0003] 1. Field

[0004] This invention generally relates to the recombinant production of therapeutic proteins. More specifically, the invention is directed to glycosylated interleukin-2 muteins which can selectively activate T cells (PHA-blasts) and reduce activation of Natural Killer (“NK”) cells.

[0005] 2. Background

[0006] The biological activity of a glycoprotein is dependent upon not only the integral structure of the protein, but also the properties of the oligosaccharide covalently attached to the protein. By influencing the physico-chemical and biological properties of proteins, oligosaccharides can modulate the therapeutic effect of a glycoprotein pharmaceutical. It is well recognized that glycosylation can affect solubility, resistance to proteolytic attack and thermal inactivation, quaternary structure, activity, targeting, antigenicity, functional activity, and half-life of the protein. The role of oligosaccharide in determining the in vivo activity of EPO and the half-life of tissue plasminogen activator has been reported.

[0007] Proleukin® (interleukin-2) has been approved by the FDA to treat melanoma and renal carcinoma. However, due to the toxic side effects associated with interleukin-2, there is a need for a less toxic IL-2 mutein that allows greater therapeutic use of this interleukin. Although non-glycosylated interleukin-2 has been produced in E. coliwith full biological activity, proper refolding of the recovered protein and the potential for altered pharmacokinetics have been areas of concern. It is known that the purification of interleukin-2 derived from E. coilrequires the use of chaotropic and toxic agents such as guanidine chloride and urea. Thus it would be advantageous to produce glycosylated IL-2 muteins in mammalian cells where the use of harsh reagents can be avoided.

[0008] U.S. Pat. No. 5,417,970 to Roskam et al. (May 23, 1995), incorporated herein by reference, discloses a wild type IL-2 preparation. The above-cited related application of Shanafelt et al. discloses IL-2 muteins and states that the muteins may be expressed in a variety of cells, including microbial, plant, and animal cells, including mammalian cells. We have now found a way to make such IL-2 muteins in glycosylated form from mammalian cells. The characterization and details for making a preferred IL-2 mutein are described below.

[0009] We have developed a method for the production of glycosylated IL-2 muteins from mammalian cells. Preferably the cell host is CHO cells, but the production can be done with other cell hosts including HKB (see U.S. patent application Ser. No. 09/209,920 to Cho filed Dec. 10, 1998, incorporated herein by reference), myeloma, and 293S cells. The production medium is preferably a chemically-defined medium free of plasma protein supplements.

[0010] This invention is illustrated with a specific glycosylated polypeptide comprising a human IL-2 mutein numbered in accordance with wild-type IL-2 wherein said human IL-2 is substituted at position 88 with arginine, whereby said mutein preferentially activates T cells over NK cells. The preferred mutein is designated IL-2N88R, using conventional terminology to describe the amino acid substitution of asparagine (N) with arginine (R) at position 88 of wild type IL-2. The nomenclature of the oligosaccharide structures is as described (Fukuda et al, 1994). Mammalian glycosylation patterns are well known and are described in Fukuda et al. (1994), incorporated herein by reference.

[0011] This mutein exhibits essentially wild-type IL-2 activity on T cells. This invention is also directed to a method of treating a patient afflicted with an IL-2 treatable condition by administering a therapeutically effective amount of a human IL-2 mutein numbered in accordance with wild-type IL-2 having PHA-blast activating activity but having reduced NK cell activating activity. This method is applicable wherein the IL-2 treatable condition is HIV, cancer, autoimmune disease, infectious disease, vaccine adjuvant in cancer vaccine and conventional vaccine therapy for immune stimulation in the elderly or otherwise immunocompromised, as well as in human SCID patients, or other therapeutic application requiring stimulation of the immune system.

[0012] The FIGURE is a schematic diagram of the IL-2N88R expression vector showing the sites of the cytomegalovirus early promoter sequence (CMVe/p), the polyadenylation signal sequence (pA), and the dihydrofolate reductase (DHFR) sequence.

[0013] Development of Stable, High-producing CHO Cell Lines That Express IL-2N88R

[0014] Stable production cell lines that secrete high quantities of the IL-2N88R mutein were developed by transfecting CHO(dhfr-) cells with the expression vector shown in FIG. 1. The vector was constructed using standard recombinant DNA techniques. The expression vector contains discrete expression cassettes for the IL-2N88R gene and the amplifiable and selectable gene DHFR (dihydrofolate reductase). The IL-2N88R gene codes for a protein having the amino acid sequence given by SEQ ID NO:1. About 1×10⁶CHO (Chinese hamster ovary) cells were transfected with 10 ug of pBC1N88R (see FIGURE) using LIPOFECTIN™ reagents (Life Technology, Bethesda, Md.) according to manufacturer's instructions. The cells were then selected in the presence of 50 nM methotrexate and grown in DME/F12 media deficient in thymidine and hypoxanthine plus 5% dialyzed fetal bovine serum. Cell populations were screened for IL-2N88R production with a commercial ELISA kit (R & D Systems). The high producing populations were further selected in media containing increasing concentrations of methotrexate (100 to 400 nM methotrexate) and screened for the production of IL-2N88R. Limiting dilution cloning was then applied to derive clones with high and stable productivity. The cloning was done in the absence of methotrexate using standard tissue culture techniques. Mammalian cell culture techniques are well known and disclosed in Freshey (1992), Mather (1998), Hu et al. (1997), and Konstantinov et al (1996), each of which are incorporated herein by reference.

[0015] Serumfree Production of IL-2N88R in a Perfusion Bioreactor

[0016] Continuous production of IL-2N88R was done by continuous perfusion fermentation. A 19-liter Wheaton fermenter was inoculated with a stable CHO cell line at 2×10⁶cells/ml and perfused at a medium exchange rate of 5 liters/day. The production medium was a DME/F12-based medium supplemented with insulin (10 ug/ml) and FeSO₄.EDTA (50 uM). The cell density was maintained at 4×10⁶cells/ml. The average daily yield of the fermenter was ˜200 mg/day. The production IL-2N88R was stably maintained for 30 days.

[0017] Carbohydrate Analysis of IL-2N88R Produced from CHO Cells

[0018] IL-2N88R produced from CHO cells was purified using standard chromatography techniques involving ion exchange, reverse phase, and size exclusion chromatography. The oligosaccharide structures of IL-2N88R were characterized using glycosidases and matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS).

[0019] IL-2N88R was analyzed either directly or after sequential digestion with sialidase, beta-galactosidases and O-glycanase on a MALDI spectrometer in linear mode. The observed molecular mass was compared with calculated molecular mass and the oligosaccharide structures were identified.

[0020] The total oligosaccharide pool was released with chemical hydrazinolysis and oligosaccharide mapping was performed by high pH anion-exchange chromatography with a CarboPac PA1 column.

[0021] IL-2N88R was found to be glycosylated only with O-linked GalNAc, GalNAc-β-Gal, and GalNAc-β-Gal-α-NeuNAc, of which monosialylated GalNAc-β-Gal was the major oligosaccharide. A minor O-glycosylation site beside the site at Thr-3 was also detected. A summary of the oligosaccharide structures found in IL-2N88R was shown in the Table.

[0022] As illustrated in the above examples, we have developed a method for the production of IL-2 muteins having a mammalian glycosylation pattern. It is thought that the method may be used to easily produce any IL-2 mutein, using a variety of mammalian cells.

[0023] The above examples are intended to illustrate the invention and it is thought variations will occur to those skilled in the art. Accordingly, it is intended that the scope of the invention should be limited only by the claims below.

[0024] Cho, M-Y (1998) U.S. patent application Ser. No. 09/209,920 filed Dec. 10, 1998.

[0025] Freshey, R. I. (ed) (1992) Animal Cell Culture: A Practical Approach, 2nd ed. IRL Press, Oxford, England.

[0026] Fukuda et al. (1994) Molecular Glycobiology, IRL Press, New York

[0027] Hu, W. S., et al. (1997) Large-scale Mammalian Cell Culture, Curr Opin Biotechnol 8: 148-153

[0028] Konstantinov, K. B., et al. (1996) Control of long-term perfusion Chinese hamster ovary cell culture by glucose auxostat, Biotechnol Prog 12: 100-109

[0029] Liu (1992) Trends in Biotechnology 10: 114-120

[0030] Mather, J. P. (1998) Laboratory Scaleup of Cell Cultures (0.5-50 liters), Methods Cell Biology 57: 219-527

[0031] Roskam et al. U.S. Pat. No. 5,417,970 (May 23, 1995)

[0032] Shanafelt et al. (1998) U.S. patent application Ser. No. 09/080,080 filed May 15, 1998.