| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 31.12.02 |
| PATENT TITLE |
Mutants of human interleukin-3 |
| PATENT ABSTRACT | Biologically active deletion and substitution mutants of hIL-3 are provided. Preferred mutants are those having one or more deletions at the N-terminus (amino acids 1-14) and/or the C-terminus (amino acids 116-133, 120-130 and/or 130-133). Preferred substitution mutants include Cys.sup.16 .fwdarw.Ala.sup.16 and/or Cys.sup.84.fwdarw.Ala.sup.84, Glu.sup.50.fwdarw.Lys.sup.50 and Lys.sup.79 .fwdarw.Glu.sup.79. These mutants can be used to formulate pharmaceutical compositions. Also disclosed are antibodies directed against specific epitopes localized between amino acids 29 and 54 |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | December 8, 1995 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED |
Cunningham BC and Wells JA. High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science. Vo. 244, pp. 1081-1085, 1989.* Yang et al., Cell (1986) 47:3-10. Dorssers et al., Gene (1987) 55:115-124. Moonen et al., Proc. Natl. Acad. Sci. (1987) 84:4428-4431. Kaushansky et al., Proc. Natl. Acad. Sci. (1989) 86:1213-1217. Gough et al., Eur. J. Biochem. (1987) 169:353-358. Kuga et al., Biochem. Biophys. Res. Commun. (1989) 159:103-111. Makino et al., Proc. Natl. Acad. Sci. (1987) 84:7841-7845. Wang et al., Science (1984) 224:1431-1433. Cohen et al., Science (1986)234:349-352. Zurawski et al., EMBO J. (1988) 7:1061-1069. Robb et al., Proc. Natl. Acad. Sci. (1988) 85:5654-5658. Collins et al., Prac. Natl. Acad. Sci. (1988) 85:7709-7713. Clark-Lewis et al., Proc. Natl. Acad. Sci. (1988) 85:7897-7901. Clark-Lewis et al., Science (1986) 231:134-139. Mosely et al., Proc. Natl. Acad. Sci. (1984) 84:4572-4576. Kaushansky et al., J. Clin. Invest. (1992) 90:1879-1888. Dunbar et al, Science (1989) 245:1493-1496 |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A human interleukin-3 antagonist having an amino acid sequence which differs from the sequence of human interleukin-3 by the replacement of one or both of Cys.sup.16 or Cys.sup.84 by another amino acid so as to disturb the disulfide bridge between Cys.sup.16 or Cys.sup.84. 2. The IL-3 antagonist of claim 1 which is a human IL-3 amino acid sequence selected from the group consisting of a polypeptide analog comprising a Cys.sup.16.fwdarw.Ala.sup.16 substitution, a polypeptide analog comprising a Cys.sup.84.fwdarw.Ala.sup.84 substitution, and a polypeptide analog comprising a Cys.sup.16 Cys.sup.84.fwdarw.Ala.sup.16 Ala.sup.84 substitution. 3. A DNA molecule comprising a DNA sequence encoding an antagonist according to claim 1. 4. A vector comprising a DNA sequence encoding a polypeptide analog according to claim 1, in combination with transcription and translation regulation sequences that enable the expression of said polypeptide analog in a suitable host cell. 5. A host cell transformed with a vector according to claim 4. 6. A method to produce an antagonist of human interleukin-3 which differs from the amino acid sequence of human interleukin-3 by replacement of one or both of Cys.sup.16 or Cys.sup.84 by another amino acid so as to disturb the disulfide bridge between Cys.sup.16 or Cys.sup.84, which method comprises culturing cells according to claim 5 under conditions suitable for producing said antagonist; and recovering the antagonist from the culture. 7. A human interleukin-3 antagonist having an amino acid sequence which differs from the sequence of human interleukin-3 by a charge reversal substitution at one or more of positions 36, 50, and 79. 8. A DNA molecule comprising a DNA sequence encoding an antagonist according to claim 7. 9. A vector comprising a DNA sequence encoding an antagonist according to claim 7, in combination with transcription and translation regulation sequences that enable the expression of said antagonist within a suitable host cell. 10. A host cell transformed with a vector according to claim 9. 11. A method to produce an antagonist of human interleukin-3 which differs from the amino acid sequence of human interleukin-3 by a charge reversal substitution at one or more of positions 36, 50, and 79, which method comprises culturing cells according to claim 10 under conditions suitable for producing said antagonist; and recovering the antagonist from the culture. 12. The interleukin-3 antagonist of claim 7 having one or more charge reversal substitutions selected from the group consisting of Asp.sup.36.fwdarw.Arg.sup.36, Glu.sup.50.fwdarw.Lys.sup.50, and Lys.sup.79.fwdarw.Glu.sup.79. 13. A human interleukin-3 antagonist having an amino acid sequence which differs from the sequence of human interleukin-3 by the substitutions Arg.sup.63 Ala.sup.64.fwdarw.Pro.sup.63 Gly.sup.64. 14. A DNA molecule comprising a DNA sequence encoding an antagonist according to claim 13. 15. A vector comprising a DNA sequence encoding an antagonist according to claim 13, in combination with transcription and translation regulation sequences that enable the expression of said antagonist within a suitable host cell. 16. A host cell transformed with a vector according to claim 15. 17. A method to produce an antagonist of human interleukin-3 which differs from the amino acid sequence of human interleukin-3 by the substitutions Arg.sup.63 Ala.sup.64.fwdarw.Pro.sup.63 Gly.sup.64, which method comprises culturing cells according to claim 16 under conditions suitable for producing said antagonist; and recovering the antagonist from the culture. 18. A method to produce a biologically active analog of human interleukin-3 wherein said analog differs from the amino acid sequence of human interleukin-3 by having deleted therefrom 4 to 18 amino acids from the C terminus of human interleukin-3, which method comprises culturing cells transformed with a vector, comprising a DNA sequence encoding a polypeptide analog of human interleukin-3 having the amino acid sequence of human interleukin-3 having deleted therefrom 4 to 18 amino acids from the C terminus of the native human interleukin-3 sequence, in combination with transcription and translation regulation sequences that enable the expression of said polypeptide analog, under conditions suitable for producing said analog; and recovering the analog from the culture. -------------------------------------------------------------------------------- |
| PATENT DESCRIPTION |
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.sup.58 and Cys.sup.105 whereas deletion of the third Cys residue (125) had no effect (Wang et al., Science 224 (1984) 1431-1433; Cohen et al., Science 234 (1986) 349-352). All substitutions resulting in a disturbance of helical folding of this protein were found to give significant reductions of biological activity. The potential receptor binding site of IL-2 has been mapped on an eleven amino acid long peptide. Individual amino acid substitutions in this region had dramatic effects (Cohen et Al., (supra); Zurawski and Zurawski, EMBO J. 7 (1988) 1061-1069). Mutational analysis further revealed that different domains of IL-2 are involved in high and low affinity binding of the IL-2 receptor complex (Robb et al., Proc. Natl. Acad. Sci. USA 85 (1988) 5654-5658; Collins et al., Proc. Natl. Acad. Sci. USA 85 (1988) 7709-7713). Modified IL-3 Clark-Lewis et Al., Science et al., (1986) 134-139, performed a functional analysis of synthetic murine IL-3 analogs. They concluded that the stable tertiary structure of the complete molecule was required for full activity. A study on the role of the disulfide bridges showed that replacement of two of the four Cys residues by Ala (Cys.sup.79, Cys.sup.140.fwdarw.Ala.sup.79, Ala.sup.140) resulted in increased activity (Clark-Lewis et al., Proc. Natl. Acad. Sci. USA 85 (1988) 7897-7901). Literature on proposed and actually performed modifications of hIL-3 is scarce. International Patent Application (PCT) WO 88/00598 discloses a Ser.sup.27.fwdarw.Pro.sup.27 replacement. (It should be noted that the numbering of amino acids in WO 88/00598 includes the signal peptide of 19 amino acids.) Furthermore, suggestions are made to replace Cys with Ser, breaking the disulfide bridge, and to replace one or more amino acids at the glycosylation sites (Asn.sup.34 cys.sup.35 Ser.sup.36 and Asn.sup.89 Ala.sup.90 Ser.sup.91). EP-A-0275598 (WO 88/04691) illustrates that Ala.sup.1 can be deleted while retaining biological activity. Some mutant hIL-3 sequences are provided, viz. two double mutants, Ala.sup.1.fwdarw.Asp.sup.1, Trp.sup.13.fwdarw.Arg.sup.13 (pGB/IL-302) and Ala.sup.1.fwdarw.Asp.sup.1, Met.sup.3.fwdarw.Thr.sup.3 ((pGB/IL-304) and one triple mutant Ala.sup.1.fwdarw.Asp.sup.1, Leu.sup.9.fwdarw.Pro.sup.9, Trp.sup.13.fwdarw.Arg.sup.13 (pGB/IL-303). WO88/05469 describes deglycosylation mutants and mutants of Arg.sup.54 Arg.sup.55 and Arg.sup.108 Arg.sup.109 Lys.sup.110 (converted to the same numbering as in EP-A-0275598). The latter are suggested in order to avoid proteolysis upon expression in Saccharomyces cerevisiae by KEX2 protease. No mutated proteins are disclosed. Glycosylation and the KEX2 protease activity are only important, in this context, upon expression in yeast. Finally, EP-A-0282185 describes various mutants that may be conformationally and antigenically neutral. To achieve this, a series of synonymous amino acid substitutions are suggested. The proposed changes are aimed at keeping the structure and charge distribution of the IL-3 molecule unaltered. However, the only actually performed mutations are Met.sup.2.fwdarw.Ile.sup.2 and Ile.sup.131.fwdarw.Leu.sup.131. It is not disclosed whether the contemplated neutralities are obtained. No known extensive mutagenesis experiments on hIL-3 have been disclosed to date. The present invention provides new classes of pharmacologically interesting compounds, viz. deletion and substitution mutants of hIL-3, showing biological activities similar and in some cases possibly antagonistic to those of hIL-3. SUMMARY OF THE INVENTION In one aspect of the invention, biologically active polypeptide analogs of human interleukin-3 (also referred to hereinafter as "hIL-3 mutants" or "muteins") are provided having a deletion of at least two amino acids. Preferred mutants are those having one or more deletions at the N-terminus (amino acids 1-14) and/or the C-terminus (amino acids 120-130 and/or 130-133). In another aspect of this invention, substitution mutants of hIL-3 are disclosed having at least one of the following substitutions: Asp.sup.21 Glu.sup.22.fwdarw.Lys.sup.21 Arg.sup.22 Asp.sup.36.fwdarw.Arg.sup.36 Glu.sup.43 Asp.sup.44.fwdarw.Lys.sup.43 Arg.sup.44 Arg.sup.54 Arg.sup.55.fwdarw.Glu.sup.54 Asp.sup.55 Asp.sup.46.fwdarw.Lys.sup.46 or Arg.sup.46 Glu.sup.50.fwdarw.Lys.sup.50 or Arg.sup.50, Glu.sup.59.fwdarw.Lys.sup.59 or Arg.sup.59 Glu.sup.59.fwdarw.Gly.sup.59 or Pro.sup.59, Arg.sup.63 Ala.sup.64.fwdarw.Pro.sup.63 Gly.sup.64 Glu.sup.75.fwdarw.Arg.sup.75 or Gly.sup.75 Lys.sup.79.fwdarw.Glu.sup.79 Arg.sup.94.fwdarw.Pro.sup.94 His.sup.98 Lys.sup.100 Asp.sup.101.fwdarw.Glu.sup.98 Asp.sup.100 Gln.sup.101 Glu.sup.106.fwdarw.Lys.sup.106 Arg.sup.108 Arg.sup.109 Lys.sup.110.fwdarw.Glu.sup.108 Asp.sup.109 Glu.sup.110, Phe.sup.113 Tyr.sup.114.fwdarw.Ala.sup.113 Thr.sup.114 Cys.sup.16.fwdarw.Ala.sup.16 Cys.sup.84.fwdarw.Ala.sup.84 Cys.sup.16 Cys.sup.84.fwdarw.Ala.sup.16 Ala.sup.84 In yet another aspect of this invention, antagonists of hIL-3 are disclosed. These antagonists are substitution mutants of hIL-3 that are more potent in receptor binding than in stimulation of DNA synthesis. More specifically, the antagonists are single or double Cys mutants. Preferably Cys is replaced by Ala (Cys.sup.16.fwdarw.Ala.sup.16, Cys.sup.16 Cys.sup.84.fwdarw.Ala.sup.16 Ala.sup.84). Other mutants having an antagonistic effect are Glu.sup.50.fwdarw.Lys.sup.50 and Lys.sup.79.fwdarw.Glu.sup.79. The polypeptides are obtained through expression of suitably modified DNA sequences. Thus, the present invention also provides suitable expression vectors and host cells compatible therewith. In yet other aspects, the invention comprises pharmaceutical compositions that include biologically active peptide analogs of hIL-3 as described above, in combination with a pharmaceutically acceptable carrier. Finally, the present invention discloses monoclonal antibodies aimed at an epitope localized between amino acids 29 and 54. Other embodiments of the subject invention are readily determined by one skilled in the art. |
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