Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
1999-07-15
2002-05-07
Salimi, Ali R. (Department: 1648)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S003000, C435S004000, C435S007210, C435S069500
Reexamination Certificate
active
06383739
ABSTRACT:
The present invention concerns the use of an “immunodeficiency-virus suppressing lymphokine (ISL)” to inhibit the replication of viruses, therapeutic compositions containing ISL or nucleic acid molecules coding therefor.
ISL activity is defined by inhibition of HIV replication on primary lymphocytes (PBMC).
It is known that certain CD8
+
cells, e.g., those of human and animal origin which, in addition to being CD8
+
are HLA-DR
+
, CD28
+
, or CD11B
−
, show activity in suppressing immunodeficiency viruses, such as HIV and SIV. This activity has been attributed to a molecule referred to as immunodeficiency virus suppressing lymphokine or “ISL”. ISL is capable of inhibiting the replication of viruses in CD4
+
cells that are infected with HIV or SIV (Ennen, Findeklee, Kurth et al (Proc. Natl. Acad. Sci. USA, Vol. 91, p. 7207-7211 (1994). However, the identity of ISL has to date been unclear. It has been known since at least 1989 (Walker, C. M., and Leighly, J. A., Immunology, Vol. 66, p. 628-630 (1989)) that there exists a soluble factor secreted by stimulated human CD8
+
T lymphocytes that down-regulates HIV replication in CD4
+
T cells. However, it has until now been impossible to establish whether this activity results from a single substance, and it has also until today even been impossible to isolate and characterize a substance with this activity although there exist a lot of publications in which methods of setting up corresponding cell cultures are described and methods of purifying such antiviral factors are suggested. Such publications are, e.g., WO 94/23058 and WO 93/0883 as well as by Mackewicz et al (Lancet, 344, p. 1671-1673 (1994)); Mackewicz et al (AIDS Research and Human Retroviruses, Vol. 8, No. 6, p. 1039-1050 (1992); Castro, Walker et al (Cellular Immunology 132, p. 246-255, (1991); Blackbourn et al (Journal of Medical Primatology No. 23, p. 343-354 (1994); Chen et al (AIDS Research and Human Retroviruses Vol. 9, No. 11, p. 1079-1086)); Kannagi et al (The Journal of Immunology, Vol. 140, No. 7, p. 2237-2242 (1988)); Joag et al (Virology 200, p. 436-446 (1994)); Walker, Moody et at (Science, Vol. 234, p. 1563-1566 (1986)); Walker, Erickson et al (Journal of Virology, Vol. 65, No. 11, p. 5921-5927 (1991)); Walker, Thomson-Honnebier et al (Cellular Immunology 137, p. 420-428 (1991)); Knuchel, Bednarik et al (Journal of Acquired Immune Deficiency Syndromes, No. 7, p. 438-446 (1994)); Ennen, Findeklee, Kurth et al (Proc. Natl. Acad. Sci. USA, Vol. 91, p. 7207-7211 (1994)) and Hsueh, Walker et al (Cellular Immunology 159, p. 271-279 (1994)).
In spite of the above-mentioned intensive investigation that has been carried out on the immunodeficiency virus-suppressing activities produced from CD8
+
cells for about seven years, the biological nature (especially the molecular structure of ISL), apart from the assumption that ISL is a protein, is completely unclear. Also unclear are:
its gene or genes if it consists of several factors;
the way in which it acts on infected and non-infected CD4
+
cells. There are preliminary indications that the action of ISL is based on a negative regulation of the transcription rate of HIV-LTR (long terminal repeat);
its mechanism of action in other infections. It may be assumed that viruses whose transcription is regulated by transcription factors that are comparable to those of HIV will also be subjected to a negative regulation by ISL;
its mechanism of action on normal and malignant cell proliferation. According to the present state of knowledge it cannot be ruled out that, similar to the interferons, an inhibitory effect on normal or malignant cell proliferation may be possible;
the fact whether ISL could regulate CD4 expression;
why in the case of HIV-infected patients a decrease in the ISL activity that is measurable in vitro occurs over time;
the fact whether ISL may also be partly responsible for the long latency period between infection and development of disease in humans infected with HIV, i.e., be positively correlated with a positive prognosis.
The problem has therefore arisen of identifying ISL and to clarify whether it represents one or several substances as well as to examine it with regard to its therapeutic action on immunodeficiency viruses and other viruses.
Cruikshank and Center (Journal of Immunology 128 (1982) 2569-2574) describe a protein called “lymphocyte chemoattractant factor” (LCF), which has a sequence quite similar to the sequence of the polypeptides of the invention. It is expressed by human lymphocytes and is a member of the group of lymphokines. After appropriate purification by gel filtration a homogeneous product was obtained with a molecular weight of approximately 56,200 which is cleaved by sodium dodecyl sulphate into monomers with a molecular weight of ca. 14,400. It was assumed that this lymphokine played a role in the formation and amplification of the delayed type of immune response (delayed type hypersensitivity reaction).
The nucleic acid sequence of LCF is described by Cruikshank, W., et al., Proc. Nat. Acad. Sci. USA 91 (1994) 5109-5113. The nucleotide sequence and the protein sequence derived therefrom are available under Accession Number M90391 at GenBank data base and are shown in SEQ ID NO:3 and SEQ ID NO:4.
From Cruikshank et al (Journal of Immunology 138, 3817-3823 (1987)) it is also known that LCF stimulates the expression of interleukin 2 (IL2) receptors and HLA-DR antigens on CD4
+
lymphocytes. LCF is therefore also referred to as growth factor. Furthermore Cruikshank et al described in the Journal of Immunology 146, 2928-2934 (1991) that LCF induces CD4-dependent intracytoplasmic signals in lymphocytes and thus concluded that these signals act as a second type of messengers. In the J. Exp. Med. 173, p. 1521-1528 (1991) Rand, Cruikshank et al additionally describe the stimulation of human eosinophils by LCF and its massive production by activated T-lymphocytes. Finally in Proc. Natl. Acad. Sci. USA, Vol. 91, p. 5109-5113 (1994) Cruikshank et al described a cloning of LCF by isolating the LCF cDNA from an expression library from mitogen-stimulated mononuclear blood cells (PBMC: peripheral blood mononuclear cells) and introduction into
E. coli
to produce biologically active recombinant LCF protein (rLCF). Recombinant LCF shows an isoelectric point of 9.0 (Center, D. M., et al., J. Lab. Clin. Med. 125 (1995) 167-171).
Cruikshank, W., et al. (Proc. Nat. Acad. Sci. USA 91 (1994) 5109-5113) describes that LCF may contribute to recruitment of eosinophils and CD4
+
mononucleic cells concomitantly in intracellular reactions. Cruikshank further suggests that LCF activity on CD4
+
cells would provide a mechanism for the accumulation of non-sensitized T cells in tissue. Its ability to prime CD4
+
T cells for IL-2 responsiveness might play a role in the specific expansion of this T cell population. In WO 94/28134 the same authors suggest to use LCF as an immunosuppressive agent or as part of an immunosuppressive therapy. However, an antiviral activity of LCF was neither described in nor obvious from these publications. To the contrary, Center, D. M., et al., (1995) (supra) conclude that LCF does amplify the inflammatory process.
SUMMARY OF THE INVENTION
The subject-matter of the invention is the identification and molecular cloning of an immuno-deficiency-virus suppressing lymphokine (ISL) and the isolation of nucleic acid molecules which encode polypeptides with ISL activity. Such polypeptides have improved properties, especially a higher activity than the polypeptide described in WO 94/28134. More specifically, the invention relates to those nucleic acid molecules which encode eukaryotic ISL, including human, monkey and other species. Specifically preferred are nucleic acid molecules which hybridize to SEQ ID NO:1 under stringent conditions as set forth below and code for a polypeptide with ISL activity. It can be shown that natural, synthetic, and recombinantly produced ISL is able to suppress the replication of
Baier Michael
Kurth Reinhard
Metzner Karin
Werner Albrecht
Arent Fox Kintner Plotkin & Kahn
Budesrepublik Deutschland Vertreten Durch Den Bundesminister
Li Bao Qun
Salimi Ali R.
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