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
1995-06-06
2001-05-29
Fitzgerald, David L. (Department: 1647)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S320100, C435S325000, C435S348000, C435S354000, C435S365100, C435S252300, C435S252310, C435S252330, C435S254110, C435S254200, C435S254210, C536S023510, C530S351000, C424S085200
Reexamination Certificate
active
06238889
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to cDNA encoding human interleukin-3 (hIL-3) and its use, inter alia, in the cloning and expression in various organisms, including microorganisms, in particular yeasts, bacteria and fungi, tissue culture cells and transgenic animals and plants.
BACKGROUND OF THE INVENTION
Hemopoiesis involves the active process of proliferation and differentation of pluripotent progenitor cells into all types of mature blood cells and some specialized tissue calls. Production of functional blood cells is regulated by specific proteins, the hemopoietic growth factors (HGFs). Some of the HGFs control maturation of a specific maturation lineage, whereas others stimulate proliferation and differentiation of progenitors along multiple pathways. Much of our knowledge of the hemopoietic differentiation process has been obtained from mouse studies in vitro and in vivo, using purified growth factors. The murine growth factor interleukin-3 (mIL-3), also termed Multi-CSF, mast cell growth factor, stem cell activating factor or several other designations, stimulates the proliferation of developmentally early, multipotent cells (CFU-S) as detected by the spleen colony assay, resulting in the production of progenitor cells along the erythroid, megacaryocyte, granulocyte/macrophage, osteoblast and several other lineages. Furthermore, mIL-3 has been implicated in replication of pluripotent stem cells, probably in synergism with other HGFs.
In recent years, several groups have succeeded in cloning mIL-3 cDNA. No results have been reported so far of identifying homologous sequences in human DNA using mIL-3 DNA as a probe. Presumably, the human gene has diverged extensively from the mIL-3 gene or has lost its function during primate evolution. However, human leukocytes were found to produce a HGF(s) which can replace mIL-3 in supporting the proliferation of murine CFU-S. Thus, the existence of a human HGF was postulated, which shares biological properties with mIL-3 and therefore could be the human homolog. Yang, Y-C, et al,
Cell
(1986) 47:3-10, dated 10 October discloses cDNA encoding a protein having IL-3 Like activity from gibbon T-cells, and retrieval of a genomic DNA which encodes the human counterpart. The sequence of a cDNA encoding human IL-3 can be deduced from the human gene sequence published by Yang et al. However, said article does neither disclose nor teach a method for isolation of a cDNA encoding human IL-3, nor was the production of hIL-3 achieved. This invention describes for the first the isolation of a cDNA comprising the entire coding sequence for human IL-3.
Human IL-3 protein has never been prepared in purified form, nor have its characteristics, other than its activity in certain in vitro proliferation assays and deduced primary structure, been disclosed. The present invention permits the recovery of purified human IL-3, and identification of its characteristics through recombinant production: from a cDNA clone.
SUMMARY OF THE INVENTION
As stated above, the present invention for the time describes the isolation of a cDNA comprising the entire coding sequence for human IL-3. The low degree of homology between the DNA sequences coding for murine and human IL-3 does not permit the retrieval of a cDNA for hIL-3 by hybridization with the mIL-3 coding sequence. Unexpectedly, the hIL-3 cDNA clone could be isolated by exploiting the rather high degree of homology in the 3′ noncoding part of the cDNA's. The availability of the cDNA clone permits the production of hIL-3 by a wide range of host organisms. Subsequent to large scale production the protein may be purified and used therapeutically.
The present invention permits production of recombinant human IL-3 protein in a wide range of host cells by transcription and translation from a cDNA sequence encoding the human IL-3 protein. The production of the protein is illustrated in several hosts, including
E. coli,
COS cells, C127 cells,
B. subtilis
and
B. licheniformis, S. cerevisiae
and
K. lactis,
hereinbelow. Production in other hosts using appropriate expression systems is also made possible by provision of the intronless cDNA. More generally, the availability of antihuman IL-3 antibodies which permit identification of colonies exhibiting successful production of the recombinant protein aids in production of human IL-3 from any recombinant system.
In one aspect, therefore, the invention is directed to a recombinant, intronless, DNA encoding human IL-3 protein.
In another aspect, it is directed to expression systems capable of effecting the expression of said DNA sequence encoding hIL-3 in an appropriate host.
In other aspects, the invention is directed to recombinant human IL-3 protein in glycosylated or unglycosylated form, to human IL-3 free of substances normally accompanying said protein, and to antibodies specifically reactive with these recombinant or purified proteins.
REFERENCES:
patent: 4675285 (1987-06-01), Clark et al.
patent: 4703008 (1987-10-01), Lin
patent: 4727138 (1988-02-01), Goeddel et al.
patent: 4810643 (1989-03-01), Souza
patent: 4877729 (1989-10-01), Clark et al.
patent: 0138133 (1985-04-01), None
patent: 0224294 (1987-06-01), None
patent: 0244042 (1987-11-01), None
patent: 8800598 (1988-01-01), None
patent: 8804691 (1988-06-01), None
Yang et al.,Cell(1986)47:3-10.
Clark-Lewis et al.,Science(1986) 231:134-139.
DeLamarter et al.,EMBO J.(1985) 4(10):2575-2581.
Dorssers et al.,Exp. Hematol. (1984) 12(6):357.
Fung et al.,Nature(1984) 307:233-237.
Garland et al.,Exp. Hematol.(1983) 11(8):757-761.
Hapel et al.,Blood(1985) 65(6):1453-1459.
Higashi et al.,J. Bio. Chem.(1983) 258(15):9522-9527.
Ihle et al.,Adv. Viral. Oncology(1984) 4:95-137.
Kinder et al.,Proc. Natl. Acad. Sci.(1986) 83:1001-1005.
Kriegler et al.,Blood(1982) 60(2):503-508.
Lemischka et al.,Cell(1985) 45:917-927.
March et al.,Nature(1985) 315:641-647.
Metcalf et al.,Blood(1986) 67(2):257-267.
Metcalf,Brit. J. Hematol.(1986) 62:409-412.
Miyatake et al.,Proc. Natl. Acad. Sci.(1985) 82:316-320.
Schrader et al.,Proc. Natl. Acad. Sci.(1986) 83:2458-2462.
Shaw et al.,Cell(1986) 46:659-667.
van Bekkum et al., Bone Marrow Transplantation: Biological Mechanisms and Clinical Practice, Marcel Dekker, Inc., New York, 1985, pp. 1-72.
Whetton et al.,TIBSMay, 1986, pp. 207-211.
Yokota et al.,Adv. Gene Technol.(1985) 2:49-52.
Yokota et al.,Proc. Natl. Acad. Sci.(1984) 91:1070-1074.
Zwarthoff et al.,Nuc. Acid Res.(1985) 13(3):791-804.
Cohen et al.,Nucleic Acids Research(1986) 14(9):3641-3658.
Parks et al.,Journal of Biological Chemistry(1989) 264(10):5420-5427.
Park et al.,J. Biol. Chem.(1989) 264(10):5420-5422.
Alberts, B. et al., Eds., “Molecular Biology of The Cell” Second Edition, (1989) Garland Publishing, Inc., New York, p. 115.
Ayala, F.J., & Kiger, V.A. (1980)Modern Genetics, Menlo Park: Benjamin/Cummings; pp. 45, 47, 48.*
Hopp, T.P. (1986)J. Immunol. Meth.88: 1-18.*
Jeong, M.C., et al. (1998)Mol. Cell. Probes12: 49-53, 1998.*
Schweiger, A., et al.J. Allergy Clin. Immunol.105 (1,2S):189, Jan. 2000.
Dorssers Lambertus Christian Johannes
Van Leen Robert Willem
Vos Yvonne Johanna
Wagemaker Gerard
DSM N.V.
Fitzgerald David L.
Morrison & Foerster / LLP
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