Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
2000-11-09
2004-10-26
Sisson, Bradley L. (Department: 1634)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S069100, C435S069500, C435S069520, C530S345000, C530S825000, C530S866000, C530S867000
Reexamination Certificate
active
06808902
ABSTRACT:
BACKGROUND OF THE INVENTION
Recombinant proteins are an emerging class of therapeutic agents. Such recombinant therapeutics have engendered advances in protein formulation and chemical modification. Such modifications can protect therapeutic proteins, primarily by blocking their exposure to proteolytic enzymes. Protein modifications may also increase the therapeutic protein's stability, circulation time, and biological activity. A review article describing protein modification and fusion proteins is Francis (1992),
Focus on Growth Factors
3:4-10 (Mediscript, London), which is hereby incorporated by reference.
One useful modification is combination with the “Fc” domain of an antibody. Antibodies comprise two functionally independent parts, a variable domain known as “Fab”, which binds antigen, and another domain known as “Fc”, which links to such effector functions as complement activation and attack by phagocytic cells. An Fc has a long serum half-life, whereas an Fab is short-lived. Capon et al. (1989),
Nature
337:525-31. When constructed together with a therapeutic protein, an Fc domain can provide longer half-life or incorporate such functions as Fc receptor binding, protein A binding, complement fixation and perhaps even placental transfer. Id. Table 1 summarizes use of Fc fusions known in the art.
TABLE 1
Fc fusion with therapeutic proteins
Fusion
Therapeutic
Form of Fc
partner
implications
Reference
IgG1
N-terminus of
Hodgkin's disease;
U.S. Pat. No.
CD30-L
anaplastic lymphoma; T-
5,480,981
cell leukemia
Murine Fc&ggr;2a
IL-10
anti-inflammatory;
Zheng et al. (1995), J.
transplant rejection
Immunol. 154: 5590-600
IgG1
TNF receptor
septic shock
Fisher et al. (1996), N.
Engl. J. Med. 334: 1697-
1702; Van Zee, K. et al.
(1996), J. Immunol. 156:
2221-30
IgG, IgA,
TNF receptor
inflammation, autoimmune
U.S. Pat. No. 5,808,029,
IgM, or IgE
disorders
issued Sept. 15, 1998
(excluding
the first
domain)
IgG1
CD4 receptor
AIDS
Capon et al. (1989),
Nature 337: 525-31
IgG1,
N-terminus
anti-cancer, antiviral
Harvill et al. (1995),
IgG3
of IL-2
Immunotech. 1: 95-105
IgG1
C-terminus of
osteoarthritis;
WO 97/23614, published
OPG
bone density
Jul. 3, 1997
IgG1
N-terminus of
anti-obesity
PCT/US 97/23183, filed
leptin
Dec. 11, 1997
Human Ig
CTLA-4
autoimmune disorders
Linsley (1991), J. Exp.
C&ggr;1
Med. 174: 561-9
Despite their advantages, use of Fc fusion molecules may be limited by misfolding upon expression in a desired cell line. Such misfolded Fc fusion molecules may generate an immune response in vivo or may cause aggregation or stability problems in production.
SUMMARY OF THE INVENTION
The present invention concerns a process by which a misfold in an Fc fusion molecule can be prevented or corrected. In one embodiment, the process comprises:
(a) preparing a fusion molecule comprising (i) a pharmacologically active domain and (ii) an Fc domain;
(b) treating the fusion molecule with a copper (II) halide; and
(c) isolating the treated fusion molecule.
The preferred copper (II) halide is CuCl
2
. The preferred concentration thereof is at least about 10 mM for fusion molecules prepared in
E. Coli
; at least about 30 mM for fusion molecules prepared in CHO cells.
An alternative embodiment of the process comprises the following steps:
(a) preparing a fusion molecule comprising (i) a pharmacologically active domain and (ii) an Fc domain;
(b) treating the fusion molecule with guanidine HCl at a concentration of at least about 4 M;
(c) increasing the pH to about 8.5; and
(d) isolating the treated fusion molecule.
Each of these processes can be employed with any number of pharmacologically active domains. Preferred pharmacologically active domains include OPG proteins, leptin proteins, TNF-&agr; inhibitors (e.g., wherein the fusion molecule is etanercept), IL-1 inhibitors (e.g., IL-1ra proteins, which are preferred), and TPO-mimetic peptides. Also within the claimed process are molecules in which the pharmacologically active compound is an antibody. The Fc domain preferably has a human sequence, with an Fc sequence derived from IgG1 most preferred. An exemplary Fc sequence is shown in
FIG. 5
hereinafter.
Although mostly contemplated as therapeutic agents, compounds of this invention may also be useful in screening for such agents. For example, one could use an Fc-peptide (e.g., Fc-SH2 domain peptide) in an assay employing anti-Fc coated plates. The vehicle, especially Fc, may make insoluble peptides soluble and thus useful in a number of assays.
The compounds prepared by the process of this invention may be used for therapeutic or prophylactic purposes by formulating them with appropriate pharmaceutical carrier materials and administering an effective amount to a patient, such as a human (or other manual) in need thereof.
Numerous additional aspects and advantages of the present invention will become apparent upon consideration of the figures and detailed description of the invention.
REFERENCES:
patent: 4572798 (1986-02-01), Koths et al.
patent: 5075222 (1991-12-01), Hannum et al.
patent: 5480981 (1996-01-01), Goodwin et al.
patent: 5654403 (1997-08-01), Smith et al.
patent: 5808029 (1998-09-01), Brockhaus et al.
patent: WO 88/08003 (1988-10-01), None
patent: WO 97/23614 (1997-07-01), None
patent: WO 97/28828 (1997-08-01), None
patent: WO 9824477 (1998-06-01), None
patent: WO 98/48024 (1998-10-01), None
Arend et al. (1985), “Effects of immune complexes on production by human monocytes of interleukin 1 or an interleukin 1 inhibitor,”J. Immunol.134(6): 3868-3875.
Balavoine et al. (1985), “Collegenase-and PGE2stimulating activity (interleukin-1 like) and inhibitor in urine from a patient with monocytic leukaemia,” Kluger M.J. Oppenheim JJ, Powanda MC, ‘eds’.The Physiological, Metabolic, and Immunologic Actions of Interleukin-1, New York: Alan R. Liss, Inc., pp. 429-436.
Deleuran et al. (1992), “Localization of interleukin-1 alpha, type 1 interleukin-1 receptor and interleukin-1 receptor antagonist in the synovial membrane and cartilage/pannus junction in rheumatoid arthritis,”Br. J. Rheumatol.31: 801-809.
Eisenberg et al. (1990), “Primary structure and functional expression from complementary DNA of a human interleukin-1 receptor antagonist,”Nature343: 341-346.
Firestein et al. (1992), “IL-1 receptor antagonist protein production and gene expression in rheumatoid arthritis and osteoarthritis synovium,”J. Immunol.149(3): 1054-1062.
Hannum et al. (1990), “Interleukin-1 receptor antagonist activity of a human interleukin-1 inhibitor,”Nature343(6256): 336-340.
Lebsack et al. (1991), “Subcutaneous IL-1 receptor antagonist in patients with rheumatoid arthritis,”Arthritis Rheum.34(suppl): S67.
Mazzei et al. (1990), “Purification and characterization of a 26-kDa competitive inhibitor of interleukin 1,”Eur. J. Immunol.20: 683-689.
Merewether et al. (2000), “Development of Disulfide Peptide Mapping and Determination of Disulfide Structure of Recombinant Human Osteoprotegerin Chimera Produced inEscherichia coli ,”Archives of Biochemistry and Biophysics375(1): 101-110.
Prieur et al. (1987), “Specific interleukin-1 inhibitor in serum and urine of children with systemic juvenille chronic arthritis,”The Lancet2: 1240-1242.
Protein Function, a practical approach (1997), p. 77, protocol 6. Ed. T.E. Creighton, Oxford University Press, Oxford.
Schwab et al. (1991), “Pro-and anti-inflammatory roles of interleukin-1 in recurrence of bacterial cell wall-induced arthritis in rats,”Infect. Immun.59(12): 4436-4442.
Seckinger et al. (1987), “A urine inhibitor of interleukin 1 activity that blocks binding,”J. Immunol.139(5): 1546-1549.
Seckinger et al. (1990), “Natural and recombinant human IL-1 receptor antagonists block the effects of IL-1 on bone resorption and prostaglandin production,”J. Immunol.145(12): 4181-4184.
Capon et al. (1989),Nature337:525-531.
Fisher et al. (1996),N. Engl. J. Med.334:1697-1702.
Harvill et al. (1995),Immunotech.1:95-105.
Linsley (1991),J. Exp. Med.174:561-569.
Van Zee et al. (1996),J. Immunol.156:2221-2230.
Zheng et al. (1995),J. Immunol.154:5590-5600.
Kosky Andrew A.
O'Conner Sheila R.
Treuheit Michael J.
Amgen Inc.
Marshall & Gerstein & Borun LLP
Sisson Bradley L.
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