Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of...
Reexamination Certificate
2000-07-07
2002-10-29
Spector, Lorraine (Department: 1646)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
C424S093100, C530S351000
Reexamination Certificate
active
06472208
ABSTRACT:
BACKGROUND OF THE INVENTION
(a) Field of the Invention
The invention relates to a novel method for producing human natural interferon-&agr; using ex vivo expanded cord blood hematopoietic cells.
(b) Description of Prior Art
Interferons (IFNs) are a class of cytokines with pleiotropic biological activities (Pfeffer, L. M. et al.,
Cancer Res,
58(12): p. 2489-99, 1998). Originally described as potent anti-viral agents, IFNs are now known to also have anti-proliferative and immunomodulatory activities. IFNs are classified as Type I and Type II depending on their structure and stability in acid medium. Type I IFNs are subclassified by homology of amino acid sequence and type of producing cells as IFN-&agr; (leukocytes), IFN-&bgr; (fibroblasts) and IFN-&ohgr; (leukocytes). Type II IFN is acid-labile and comprises only IFN-&ggr; produced by activated T cells and NK cells. In opposition to IFN-&bgr; and IFN-&ggr;, IFN-&agr; is molecularly heterogeneous and comprises at least 13 proteins coded by more than 14 genes (Pfeffer, L. M.,
Semin Oncol,
24(3 Suppl 9): p. S9-63-S9-69, 1997). The similarity between the IFN-&agr; proteins is between 78% and 95% at the protein level and 79/166 amino acids are conserved in the family. Furthermore allelic forms of IFN-&agr; genes with variations in 1 to 4 amino acids have been described thus greatly increasing the number of potential IFN-&agr; proteins (Nyman, T. A., et al.,
Biochem J,
329(Pt 2): p. 295-302, 1998). The exact reason for the existence of so many IFN-&agr; species remains unclear since all species exhibit the same biological activities. However the specific activities of each species in different biological assays vary greatly by a factor up to 1000-fold and this observation could be related in some cases to differences in affinity of the various IFN-&agr; species for the receptor (IFN-&agr;R) expressed on various cells (Pfeffer, L. M.,
Semin Oncol,
24(3 Suppl 9): p. S9-63-S9-69, 1997). It remains to be seen if the mixture of IFN-&agr; species varies according to the producing cells or the inducing agent.
In the 1970s, the anti-proliferative activity of Type I IFNs attracted much interest for potential use in cancer treatment. At that time, the available IFN-&agr; was natural (nIFN-&agr;) and produced mainly by overnight culture of pooled human blood leukocytes after infection with Sendai virus following protocols developed by the group of Cantell in Helsinki (Cantell, K., S., et al.,
Methods Enzymol,
78(Pt): p. 29-38, 1981). This preparation of nIFN-&agr; has been recently shown to contain at least 9 of the known IFN-&agr; species (Nyman, T. A., et al.,
Biochem J,
329(Pt 2): p. 295-302, 1998). The molecular cloning of the first IFN-&agr; cDNA (species 2a) in 1979 shifted the interest to recombinant molecules (rIFN-&agr;) produced in bacteria because of the possibility of large supply which was difficult to achieve with the leukocyte-derived nIFN-&agr;. In 1987, the first rIFN-&agr; was approved by the FDA for use in the treatment of hairy cell leukemia. This rIFN-&agr;2a molecule was followed shortly after by a rIFN-&agr;2b species (Baron, S., et al.,
Journal of American Medicine Association,
266(10), p. 1375-1383, 1991). Today the rIFN-&agr;s are widely used in the treatment of more than 10 malignancies and virologic diseases including the widespread hepatitis B and C infections (Pfeffer, L. M. et al.,
Cancer Res,
58(12): p. 2489-99, 1998). However therapeutic use rIFN-&agr; results in clinical improvement in only a fraction of the patient populations. For example, the rIFN-&agr; treatment of Hepatitis C-infected patients is highly effective only in 30% of the cases. Significant side effects of rIFN-&agr; injection are routinely observed and may prevent the long-term treatment necessary to eradicate the virus. Also a significant proportion of IFN-&agr;-treated patients (10-20%) develop antibody inhibitors which may interfere with the therapeutic effect or prevent continuous treatment. These limitations and side effects and the fact that the two available rIFN-&agr; species (2a and 2b) may not be the most effective IFN-&agr; species in some diseases have renewed the interest in the nIFN-&agr; preparations. Indeed less side effects and frequency of antibody inhibitors formation have been observed in some small scale clinical trials. Also the switch from rIFN-&agr; to nIFN-&agr; could permit to prolong the treatment of patients which have developed a resistance to rIFN-&agr;. With the same objectives, a synthetic rIFN-&agr; termed rIFN-&agr;-con1 has been designed in vitro by assigning at each position in the primary sequence, the amino acid most frequently observed in several IFN-&agr; species. The rIFN-&agr;-con1 is also tested in clinical trials.
The nIFN-&agr; is currently produced by overnight culture of Sendai virus-infected human leukocytes isolated from the buffy coats prepared from several hundred blood donations (Cantell, K., S., et al.,
Methods Enzymol,
78(Pt): p. 29-38, 1981). This procedure has several limitations. On one hand, it requires tight logistics with the blood bank since production of nIFN-&agr; must be done with fresh cells and initiated within 24 hours of blood collection. In this regard, the increasing use of pre-storage leukodepletion by filtration to reduce contamination of red blood cells and platelets by leukocytes will further increase the difficulties in recovering leukocytes for nIFN-&agr; production. On the other hand, lots of nIFN-&agr; must be prepared from pools of leukocytes prepared from thousands of blood donations. Although the nIFN-&agr; can be highly purified and subjected to viral inactivation procedures, there are concerns about possible contamination of the final product with untested or unknown infectious agents.
Much work has been done to characterize the IFN-&agr; producing cells present in the peripheral blood. Early results showed that the major IFN-&agr; producing cells constituted only a minor portion of blood leukocytes (Feldman, S. B., et al.,
Virology,
204(1): p. 1-7, 1994; and Brandt, E. R., et al.,
Br J Haematol,
86(4): p. 717-725, 1994). Subsequent work showed that these cells possessed markers characteristic of immature monocyte/dendritic cells (Eloranta, M. L., et al.,
Scand J Immunol,
46(3): p. 235-241, 1997; and Svensson, H., et al.,
Scand J Immunol,
44(2): p. 164-172 1996). Recently a major IFN-&agr;-producing cell in blood was isolated and shown to have markers characteristics of lymphoid dendritic cells (DC) precursors (CD4+CDllc−) (Siegal, F. P., et al.,
Science,
284(5421): p. 1835-1837, 1999). Dendritic cells are terminally differentiated lymphoid and myeloid cells that have important immunomodulatory roles in antigen presentation and cytokine secretion. Mature DCs are constantly produced from both lymphoid and myeloid precursors (Hart, D. N.,
Blood,
90 (9): p. 3245-3287, 1997). One strategy to increase the nIFN-&agr; productivity of blood leukocytes would be to expand the IFN-&agr;-producing cells in vitro prior to IFN-&agr; induction with Sendai virus. Culture conditions (GM−CSF+IL4) that permits to expand the blood DCs have been described but the expansion factor remained limited (10-20×) (Romani, N., et al.,
J Exp Med,
180(1): p. 83-93, 1994). Also the presence of cytotoxic T lymphocytes would prevent the pooling of the leukocytes from different donors for the culture expansion phase. However hematopoietic stem cells (HSCs) can now be expanded in vitro for several weeks (Piacibello, W., et al.,
Blood,
89(8): p. 2644-2653, 1997; Traycoff, C. M., et al.,
Exp Hematol,
26 (1): p. 53-62, 1998; and Ziegler, B. L. and L. Kanz,
Curr Opin Hematol,
5(6): p. 434-440, 1998). In these cultures, the HSCs proliferate and differentiate autonomously into progenitors of the various blood cell lineages. But in most instances, differentiation of HSCs is not complete and does not proceed to the mature blood cell stage in these cultures (Ziegler, B. L. and L. Kanz,
Curr Opin Hematol,
5(6): p. 434-440, 1998).
It would be
Lemieux Real
Néron Sonia
Proulx Chantal
Héma-Québec
Jiang Dong
Nixon & Peabody LLP
Spector Lorraine
LandOfFree
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