Antagonists of interleukin-15

Details Antagonists of interleukin-15 Antagonists of interleukin-15

1998-08-14

2001-01-02

Navarro, Albert (Department: 1645)

Drug, bio-affecting and body treating compositions

Lymphokine

C424S085200, C530S300000, C530S350000

Utility Patent

active

06168783

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates generally to antagonists of a mammalian epithelium-derived T-cell factor polypeptide referred to herein as interleukin-15 (“IL-15”). It more particularly relates to muteins of IL-15, monoclonal antibodies against IL-15 and IL-15 conjugates that each significantly reduce the ability of IL-15 to stimulate the proliferation of T-lymphocytes in an in vitro CTLL assay. Also included in the invention are methods for treating various disease states in mammals where a reduction in IL-15 activity is desired.
BACKGROUND OF THE INVENTION
Interleukin-15 is a known T-cell growth factor that can support proliferation of an IL-2-dependent cell line, CTLL-2. IL-15 was first reported by Grabstein et al., in
Science,
264:965 (1994) as a secreted cytokine comprising a 162-amino acid precursor polypeptide that contains a 48-amino acid leader sequence that results in a 114-amino acid mature protein. Grabstein et al. also describe the cloning of the full-length human cDNA encoding the 162-amnino acid precursor, which contains a 316 bp 5' noncoding region and a 486 bp open reading frame (or a 489 bp open reading frame when including the 3 bp for the stop codon) and a 400 bp 3' noncoding region.
IL-15 shares many properties with IL-2. These properties include proliferation and activation of human and murine T cells, the induction of lymphokine activated killer cell (LAK) activity, natural killer cell (NK) activity, and cytotoxic T lymphocytes (CTL) activity, and costimulation of B cell proliferation and differentiation.
Additionally, IL-15 and IL-2 are structurally homologous molecules that are able to bind to at least three distinct receptor subunits on the T cell membrane surface. IL-2 receptors contain at least three subunits, &agr;, &bgr; and &ggr; (Toshikazu et al.,
Science,
257:379 (1992)). Both IL-15 and IL-2 share binding to a common &bgr;-&ggr; subunit complex, while each of IL-15 and IL-2 bind to a specific &agr;-receptor subunit (IL-15R&agr; and IL-2R&agr;, respectively). Recently, the IL-15R&agr; was discovered and is the subject of copending application U.S. Ser. No. 08/300,903, now U.S. Pat. No. 5,591,630. Antibodies directed against the &agr;-chain of the IL-2 receptor (anti-IL-2R&agr;) have no effect on IL-15 binding (Grabstein et al., Id.). Antibodies directed against the &bgr;-subunit of the IL-2 receptor, i.e., TU27, TU11, or Mik&bgr;1, however, are able to block the activity of IL-15, suggesting that IL-15 uses the &bgr;-subunit for signaling. Similarly, the &ggr;-chain of the IL-2 receptor is required for signal transduction (Giri et al.,
EMBO J.,
13:2822 (1994)). The combination of the &bgr; and the &ggr;-subunits of the IL-15 receptor complex, but neither subunit alone, bound IL-15 on transfected COS cells.
Certain disease states and physiological conditions are mediated by T cells. Such diseases include organ transplant rejection, graft versus host disease, autoimmune disease, rheumatoid arthritis, inflammatory bowel disease, dermatologic disorders, insulin-dependent diabetes mellitus, ocular disorders and idiopathic nephrotic syndrome/idiopathic membranous nephropathy. Indeed, allograft rejection and graft-versus-host disease (GVHD) have been associated with increased IL-2 receptor expression. T cells activated in response to foreign histocompatibility antigens appear to express the IL-2 receptor complex. Various therapies have been proposed and studied. For example, Tinubu et al. (
J. Immunol.,
153:4330 (1994)), reported that the anti-IL-2R&bgr; monoclonal antibody, Mik&bgr;1, prolongs primate cardiac allograft survival. There is an increase in IL-2R&bgr;-subunit expression on CD4- and CD8-expressing cells in association with acute allograft rejection, which indicates that the IL-2R&bgr;-subunit expression seems to increase on alloreactive T cells. See, for example, Niguma et al.,
Transplantation,
52:296 (1991).
However, prior to the present invention, there have been no therapies that focused on the IL-15 ligand-receptor interaction as a means of treating GVHD or in promoting allograft survival.
SUMMARY OF THE INVENTION
The invention is directed to IL-15 antagonists and a method of using the antagonists for treatment of human disease. In particular, such treatment includes promoting allograft survival in mammals and treating GVHD. The IL-15 antagonists are effective by preventing IL-15 from transducing a signal to a cell through either the &bgr;- or &ggr;-subunits of the IL-15 receptor complex, thereby antagonizing IL-15's biological activity. Certain of the antagonists according to the invention may interfere with the binding of IL-15 to the &bgr;- or &ggr;-subunits of the IL-15 receptor complex, while not substantially interfering with the binding of IL-15 to IL-15R&agr;.
Antagonists according to the invention include muteins of mature, or native, IL-15, wherein IL-15 has been mutagenized at one or more amino acid residues or regions that play a role in binding to the &bgr;- or &ggr;-subunit of the IL-15 receptor complex. Such muteins prevent IL-15 from transducing a signal to the cells through either of the &bgr;- or &ggr;-subunits of the IL-15 receptor complex, while maintaining the high affinity of IL-15 for the IL-15R&agr;. Typically, such muteins are created by additions, deletions or substitutions at key positions, for example, Asp
56
or Gln
156
of simian and human IL-15 as shown in SEQ ID NOS: 1 and 2, respectively. It is believed that the Asp
56
affects binding with the &bgr;-subunit and that the Gln
156
affects binding with the &ggr;-subunit of the IL-15 receptor complex.
In addition, the invention encompasses monoclonal antibodies that immunoreact with mature. IL-15 and prevent signal transduction to the IL-15 receptor complex.
Further included in the scope of the invention are modified IL-15 molecules that retain the ability to bind to the IL-15R&agr;, but have substantially diminished or no affinity for the &bgr;- and/or &ggr;-subunits of the IL-15 receptor complex. Modified IL-15 molecules can take any form as long as the modifications are made in such a manner as to interfere with or prevent binding, usually by modification at or near the target binding site. Examples of such modified IL-15 molecules include mature IL-15 or a mutein of IL-15 that is covalently conjugated to one or more chemical groups that sterically interfere with the IL-15/IL-15 receptor binding. For example, mature IL-15 may contain site-specific glycosylation or may be covalently bound to groups such as polyethylene glycol (PEG), monomethoxyPEG (mPEG), dextran, polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), poly amino acids such as poly-L-lysine or polyhistidine, albumin, gelatin at specific sites on the IL-15 molecule that can interfere with binding of IL-15 to the &bgr;- or &ggr;-chains of the IL-15 receptor complex, while maintaining the high affinity of IL-15 for the IL-15R&agr;. By taking advantage of the steric hindrance properties of the group, binding to specific receptor subunits can be antagonized. Other advantages of conjugating chains of PEG to proteins such as IL-2, GM-CSF, asparaginase, immunoglobulins, hemoglobin, and others are known in the art. For example, it is known that PEG prolongs circulation half-lives in vivo (see, Delgado, et al.,
Crit. Rev. Ther. Drug Carr. Syst.,
9:249 (1992)), enhances solubility (see, Katre, et al.,
Proc. Natl. Acad. Sci.,
84:1487 (1987)) and reduces immunogenicity (see, Katre, N. V.,
Immunol.
144:209 (1990)).
The invention also is directed to the use of the antagonists in a method of treating a disease or condition in which a reduction in IL-15 activity on T cells is desired. Such diseases include organ transplant rejection, graft versus host disease, autoimmune disease, rheumatoid arthritis, inflammatory bowel disease, dermatologic disorders, insulin-dependent diabetes mellitus, ocular disorders and idiopathic nephrotic syndrome/idiopathic membranous nephropathy. In particular, in allograft rejection, IL-15 activity may lead to a host immune respo

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