Drug – bio-affecting and body treating compositions – Lymphokine – Interleukin
Reexamination Certificate
1995-12-08
2002-12-31
Kunz, Gary L. (Department: 1647)
Drug, bio-affecting and body treating compositions
Lymphokine
Interleukin
C530S351000, C536S023500, C435S320100, C435S325000, C435S252300, C435S254110, C435S069520
Reexamination Certificate
active
06500417
ABSTRACT:
TECHNICAL FIELD
The present invention relates to mutants of colony stimulating factors, obtained by recombinant DNA techniques. More specifically, the invention relates to mutants of interleukin-3, containing one or more deletions and/or one or more substitutions, with interesting pharmacological properties.
BACKGROUND OF THE INVENTION
Historically, factors affecting hematopoietic cells have been detected in an assay measuring the proliferation and/or differentiation of bone marrow cells in soft agar cultures. The factors showing this activity have been collectively called colony-stimulating factors (CSFs). More recently, it has been found that a variety of CSFs exist which, in part, can be classified by the hematopoietic lineages that are stimulated.
In human and murine systems, these proteins include G-CSF and M-CSF. These proteins stimulate the in vitro formation of predominantly neutrophilic granulocyte and macrophage colonies, respectively. Interleukin-2 (“IL-2”) stimulates the proliferation of both activated T-cells and activated B-cells, but is not considered a colony stimulating factor.
GM-CSF and interleukin-3 (“IL-3”, also known as “Multi-CSF”) stimulate the formation of macrophage and both neutrophilic and eosinophilic granulocyte colonies. In addition, IL-3 stimulates the formation of mast, megakaryocyte and pure and mixed erythroid colonies (D. Metcalf, “The hematopoietic colony-stimulating factors”, 1984, Elsevier, Amsterdam, and D. Metcalf, Science 299 (1985) 16-22).
Growth factor-induced cell proliferation is a complicated process. Following highly specific binding of the growth factor to its receptor at the cell surface, the complex is internalized by endocytosis and induces an intracellular response often preceded by phosphorylation of the receptor (Sibley e al., Cell 48 (1987) 913-922). These intracellular signals result in specific gene transcription and finally in DNA synthesis and cell replication.
There is considerable interest in the CSFs, since they may be therapeutically useful for restoring depressed levels of hematopoietic and lymphoid stem cell-derived cells.
Human IL-3 (“hIL-3”) is such a CSF. Mature hIL-3 consists of 133 amino acids; the protein contains one disulfide bridge and has two potential glycosylation sites (Yang et al., Cell 47 (1986) 3-10). It has inter alia the following activities:
1) stimulation of colony formation by human hematopoietic progenitor cells wherein the colonies formed include erythroids, granulocytes, megakaryocytes, granulocyte macrophages, and mixtures thereof; and
2) stimulation of DNA synthesis by human acute myelogenous leukemia (AML) blasts.
Useful agonists and antagonists of a proteir can be created once the structure-function relationship of the molecule is understood. Generally, this relationship is studied by modifying, replacing or deleting amino acids. In this way, information can be obtained about the importance of each of the amino acids for the activity of the protein. Important domains of proteins may be the active site, metal and cofactor binding sites, receptor binding sites, the amino acids involved in subunit interactions, and the antigenic determinants.
Once the primary sequence of a protein has been determined, various procedures can be employed to study the above-mentioned characteristics. For example, if primary structures of homologous proteins from other species are available, the sequences can be compared. Conserved sequences are often indicative of the importance of certain amino acids.
Secondary structures can be predicted with the use of known algorithms. See, e.g., Hopp and Woods, Proc. Natl. Acad. Sci. USA 78 (1981) 3824-3828, Garnier et al., J. Mol. Biol. 120 (1978) 97-120, Biou et al., Prot. Eng. 2 (1988) 185-191, Carmenes et al., Biochem. Biophys. Res. Commun. 159 (1989) 687-693.
If interspecies homology between homologous proteins is high and the 3-D structure of one of them is known, important amino acids can also be deduced from this structure.
Primary and/or spatial-structure data can be used to make an educated guess for mutagenesis experiments. Expression of mutagenized proteins and the testing of these muteins in biological assays provides information about the relative importance of certain amino acids.
The aim of the present invention is to provide IL-3 mutants with similar or improved pharmaceutical properties with respect to the native IL-3, preferably using the procedures mentioned above.
BACKGROUND LITERATURE
Human Interleukin-3
In 1984, cDNA clones coding for murine IL-3 were isolated (Fung et al., Nature 307 (1984) 233-237 and Yokota et al., Proc. Natl. Acad. Sci. USA 81 (1984) 1070-1074). This CDNA would not hybridize with human DNA or cDNA clones. Thus, it was speculated that a human counterpart for murine IL-3 (mIL-3) did not exist. This belief was reinforced by the wide spectrum of activities of the human GM-CSF. Finally, in 1986, a gibbon cDNA expression library provided the gibbon IL-3 sequence. This sequence was subsequently used as a probe against a human genomic library. This provided evidence for the presence of IL-3 in human beings (Yang et al., Cell 47 (1986) 3-10).
Meanwhile, Dorssers et al., Gene 55 (1987) 115-124, found a clone from a human cDNA library that surprisingly hybridized with mIL-3. This hybridization was the result of the high degree of homology between the 3′ noncoding regions of mIL-3 and hIL-3.
Modified CSFs (other than IL-3)
Moonen e al., Proc. Natl. Acad. Sci. USA 84 (1987) 4428-4431 describe the production of human GM-CSF by several recombinant sources including
E. coli
, yeast and animal cells. Partially purified expression products from yeast and animal cells were assayed for the effect of deglycosylation. The immunoreactivity was increased 4- to 8-fold upon removal of the N-linked oligosaccharides. The specific biological activity was increased by a factor of 20, both in the chronic myelogenous leukemia (CML) and in the human bone marrow assay.
Kaushansky e a., Proc. Natl. Acad. Sci. USA 86 (1989) 1213-1217, tried to define the region(s) of the GM-CSF polypeptide required for biological activity. Since human and murine GM-CSF do not cross-react in their respective colony-forming assays, the approach was based on the use of hybrid DNA molecules containing various lengths of the coding regions for h- and mGM-CSF. After expression in COS cells, the hybrid proteins were tested in both human and murine colony-forming assays. Two regions of GM-CSF were found to be critical for hematopoietic function. These regions are structurally characterized by an amphiphilic helix and by a disulfide-bonded loop.
Gough et al., Eur. J. Biochem. 169 (1987) 353-358, describe internal deletion mutants of murine GM-CSF. None of the mutants is reported to show biological activity.
Kuga et al., Biochem. Biophys. Res. Com. 159 (1989) 103-111, describe mutagenesis of human G-CSF. The results indicate that most of the expression products with mutations localized in the internal or C-terminal regions abolish hG-CSF activity. On the other hand, N-terminal deletion mutants missing 4, 5, 7 or 11 amino acids, out of a total of 174 amino acids, retained activity. Some of the N-terminal amino acid mutants showed increased activity.
Deletion mutants of human interleukin-1 (IL-1) have been created using available endonuclease restriction sites and expression in eukaryotic cells. The carboxyl terminal third (63 amino acids) of the polypeptide contains the active site (Makino et al., Proc. Natl. Acad. Sci. USA 84 (1987) 7841-7845). A recent study on IL-1alpha and IL-1beta shows that 140 and 147 amino acids, respectively (out of a total of 153 amino acids), are required for full biological activity (Mosley et al., Proc. Natl. Acad. Sci. USA 84 (1987) 4572-4576). Single amino acid changes at both termini result in significant decrease of biological activity. However, no detailed information with respect to the receptor-binding domain of IL-1 has been obtained from these studies.
Activity of human interleukin-2 was shown to be severely inhibited by removal of both Cys
5
Dorssers Lambertus Christiaan Johannes
van Leen Robert Willem
DSM N.V.
Kunz Gary L.
Landsman Robert S.
Morrison & Foerster / LLP
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