Human Interleukin-3(Il-3) variants and their use to promote or a

Details Human Interleukin-3(Il-3) variants and their use to promote or a Human Interleukin-3(Il-3) variants and their use to promote or a

1994-06-20

1997-01-07

Fitzgerald, David L.

Drug, bio-affecting and body treating compositions

Lymphokine

Interleukin

530351, 435 6952, A61K 3821, C07K 1454, C12N 1524

Patent

active

055914276

DESCRIPTION:

BRIEF SUMMARY
This invention relates to variants and mutants of human interleukin-3 (hIL-3), and in particular it relates to hIL-3 va sequence of wild-type hIL-3 is varied in order to obtain useful changes in activity, particularly in binding to IL-3 receptors and in biological function.
Human IL-3 is a T-cell-derived glycoprotein of Mr 23-30 kd which promotes the proliferation and differentiation of haemopoietic progenitor cells, including megakaryocytes, eosinophils, basophils, neutrophils, monocytes and T-lymphocytes. It also induces the growth and the functional activation of more mature cells, including eosinophils, basophils and monocytes. The cDNA of hIL-3 has been cloned, and the mature protein of 133 amino acids has been produced in recombinant form. The human IL-3 receptor comprises at least two components, an a chain which binds IL-3 with low affinity only, and a .beta. chain which allows high affinity binding when co-expressed with the .alpha. chain (Kitamura T, Sato N, Arak K-I and Miyajima A, 1991, Cell 66, 1165-1174).
Subsequent structure-activity relationship studies of hIL-3 have been performed by functional analysis of hIL-3 deletion and substitution variants (Lokker et al, 1991a, J. Biol. Chem. 266, 10624-10631; 1991b, EMBO J. 10, 2125-2131) using recombinant hIL-3 variants generated by site-directed mutagenesis and expression in Escherichia coli. In this work, the variants were analysed for their ability to bind to the IL-3 receptor and to induce the proliferation of the human IL-3 -dependent cell line M-07. These studies initially showed that hIL-3 residues Pro 33 and Leu 34 are essential for modulating the biological activity of hIL-3, and that certain substitution variants at residues 33 and 34, particularly the variant in which Pro 33 was substituted with Gly (Gly 33), showed an enhanced proliferation activity without a significant modification in its receptor binding capacity (Lokker et al, 1991a supra). Subsequent studies which extended the structure-activity- relationship studies showed that the hIL-3 residue Leu 111, and possibly also Lys 110, form part of an active site. Thus, substitution of Lys 110 with either Glu or Ala resulted in variants with substantially reduced activity in receptor binding and proliferation assays. Similarly, variants where Leu 111 was substituted by Pro or Met were totally inactive in these assays (Lokker et al, 1991b supra).
It has now been discovered that variants or mutants of hIL-3 in which one or more amino acids in or adjacent to the predicted "D" or fourth predicted .alpha.-helix of hIL-3 is/are replaced with another amino acid show enhanced biological activity when compared with wild-type hIL-3. This enhanced biological activity is paralleled by enhanced binding to the specific a chain of the IL-3 receptor, and suggests that the variants or mutants may be used as therapeutic agents.
According to a first aspect of the present invention, there is provided a human IL-3 variant or mutant, characterised in that one or more amino acids in or adjacent to the predicted "D" or fourth predicted .alpha.-helix of hIL-3 is/are replaced by another amino acid.
In one embodiment of this aspect of the invention, there is provided a human IL-3 variant or mutant, characterised in that amino acid 101 (Asp) and/or amino acid 116 (Lys) is/are replaced by another amino acid.
Particularly preferred variants or mutants in accordance with this aspect of the invention are:
In addition, it has also been found that replacement of one or more amino acids in the predicted "A" or first predicted .alpha.-helix with another amino acid, particularly replacement of amino acids 21, 22 and 25, results in loss of IL-3 activity to high affinity IL-3 receptors indicating that these residues form part of another IL-3 active part. It has, however, been shown that these biologically inactive mutants still retain binding ability to the .alpha. chain of the IL-3 receptor. The loss of biological activity suggests that these mutants may be used as antagonists.
According to a second aspect of this inventi

REFERENCES:
Burger, H., et al. (1990) Blood 76(11):2229-34.

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