IFNAR2/IFN complex

Details IFNAR2/IFN complex IFNAR2/IFN complex

1998-12-18

2002-04-16

Eyler, Yvonne (Department: 1646)

Drug, bio-affecting and body treating compositions

Lymphokine

Interferon

C424S085600, C424S085700, C514S002600, C435S069510, C536S023520

Reexamination Certificate

active

06372207

ABSTRACT:

FIELD OF INVENTION
The present invention relates to a Type I interferon complex, composed of the polypeptide sequence of the human interferon &agr;/&bgr; receptor (IFNAR2) extracellular domain and a Type I interferon (IFN&agr;, IFN&bgr;, and IFN&ohgr;). Such a complex improves the stability, enhances the potency, and prolongs the pharmacokinetics in vivo of free IFN for anti-viral, anti-cancer and immune modulating activity. More particularly, the complex is a fusion protein, or a covalent complex, or a non-covalent complex containing the polypeptide sequence of the entire extracellular domain of IFNAR2, or any interferon-binding subfraction thereof, complexed to a Type I interferon (IFN&agr;, IFN&bgr;, IFN&ohgr;), or any biologically active subfraction thereof.
BACKGROUND OF INVENTION
Interferons are classified either as the leukocyte and fibroblast derived Type I interferons, or as the mitogen induced or “immune” Type II interferons (Pestka et al, 1987). Through analysis of sequence identities and common biological activities, Type I interferons include interferon alpha (IFN&agr;), interferon beta (IFN&bgr;) and interferon omega (IFN&ohgr;), while Type II interferon includes interferon gamma (IFN&ggr;). The IFN&agr;, IFN&bgr; and IFN&ohgr; genes are clustered on the short arm of chromosome 9 (Lengyl, 1982). There are at least 25 non-allelic IFN&agr; genes, 6 non-allelic IFN&ohgr; genes and a single IFN&bgr; gene. All are believed to have evolved from a single common ancestral gene. Within species, IFN&agr; genes share at least 80% sequence identity with each other. The IFN&bgr; gene shares approximately 50% sequence identity with IFN&agr;; and the IFN&ohgr; gene shares 70% homology with IFN&agr; (Weissman et al, 1986; Dron et al, 1992). IFN&agr; has a molecular weight range of 17-23 kDa (165-166 amino acids), IFN&bgr;, ~23 kDa (166 amino acids) and IFN&ohgr;, ~24 kDa (172 amino acids).
Type I interferons are pleiotropic cytokines having activity in host defense against viral and parasitic infections, as anti-cancer cytokines and as immune modulators (Baron et al, 1994; Baron et al, 1991). Type I interferon physiological responses include anti-proliferative activity on normal and transformed cells; stimulation of cytotoxic activity in lymphocytes, natural killer cells and phagocytic cells; modulation of cellular differentiation; stimulation of expression of class I MHC antigens; inhibition of class II MHC; and modulation of a variety of cell surface receptors. Under normal physiological conditions, IFN&agr; and IFN&bgr; (IFN&agr;/&bgr;) are secreted constitutively by most human cells at low levels with expression being up-regulated by addition of a variety of inducers, including infectious agents (viruses, bacteria, mycoplasma and protozoa), dsRNA, and cytokines (M-CSF, IL-1&agr;, IL-2, TNF&agr;). The actions of Type I interferon in vivo can be monitored using the surrogate markers, neopterin, 2′, 5′ oligoadenylate synthetase, and &bgr;2 microglobulin (Alam et al, 1997; Fierlbeck et al, 1996; Salmon et al, 1996).
Type I interferons IFN&agr;/&bgr;/&ohgr;) act through a cell surface receptor complex to induce specific biologic effects, such as anti-viral, anti-tumor, and immune modulatory activity. The Type I IFN receptor (IFNAR) is a hetero-multimeric receptor complex composed of at least two different polypeptide chains (Colamonici et al, 1992; Colamonici et al, 1993; Platanias et al, 1993). The genes for these chains are found on chromosome 21, and their proteins are expressed on the surface of most cells (Tan et al, 1973). The receptor chains were originally designated alpha and beta because of their ability to be recognized by the monoclonal antibodies IFN&agr;R3 and IFNaR&bgr;1, respectively. Most recently, these have been renamed IFNAR1 for the alpha subunit and IFNAR2 for the beta subunit. In most cells, IFNAR1 (alpha chain, Uze subunit) (Uze et al, 1990) has a molecular weight of 100-130 kDa, while IFNAR2 (beta chain, B
L
, IFN&agr;/&bgr;R) has a molecular weight of 100 kDa. In certain cell types (monocytic cell lines and normal bone marrow cells) an alternate receptor complex has been identified, where the IFNAR2 subunit (&bgr;
S
) is expressed as a truncated receptor with a molecular weight of 51 kDa. The IFNAR1 and IFNAR2 &bgr;
S
and &bgr;
L
subunits have been cloned (Novick et al, 1994; Domanski et al, 1995). The IFNAR2 &bgr;
S
and &bgr;
L
subunits have identical extracellular and transmembrane domains; however, in the cytoplasmic domain they only share identity in the first 15 amino acids. The IFNAR2 subunit alone is able to bind IFN&agr;/&bgr;, while the IFNAR1 subunit is unable to bind IFN&agr;/&bgr;. When the human IFNAR1 receptor subunit alone was transfected into murine L-929 fibroblasts, no human IFN&agr;s except IFN&agr;8/IFN&agr;B were able to bind to the cells (Uze et al, 1990). The human IFNAR2 subunit, transfected into L cells in the absence of the human IFNAR1 subunit, bind human IFN&agr;2, binding with a Kd of approximately 0.45 nM. When human IFNAR2 subunits were transfected in the presence of the human IFNAR1 subunit, high affinity binding could be shown with a Kd of 0.026-0.114 nM (Novick et al, 1994; Domanski et al, 1995). It is estimated that from 500-20,000 high affinity and 2,000-100,000 low affinity IFN binding sites exist on most cells. Although the IFNAR1/2 complex (&agr;/&bgr;
s
or &agr;/&bgr;
L
) subunits bind IFN&agr; with high affinity, only the &agr;/&bgr;
L
pair appears to be a functional signaling receptor.
Transfection of the IFNAR1 and the IFNAR2 &bgr;
L
subunits into mouse L-929 cells, followed by incubation with IFN&agr;2, induces an anti-viral state, initiates intracellular protein phosphorylation, and causes the activation of intracellular kinases (Jak1 and Tyk2) and transcription factors (STAT 1, 2, and 3) (Novick et al, 1994; Domanski et al, 1995). In a corresponding experiment, transfection of the IFNAR2 &bgr;
S
subunit was unable to initiate a similar response. Thus, the IFNAR2 &bgr;
L
subunit is required for functional activity (anti-viral response) with maximal induction occurring in association with the IFNAR1 subunit.
In addition to membrane bound cell surface IFNAR forms, a soluble IFNAR has been identified in both human urine and serum (Novick et al, 1994; Novick et al, 1995; Novick et al, 1992; Lutfalla et al, 1995). The soluble IFNAR isolated from serum has an apparent molecular weight of 55 kDa on SDS-PAGE, while the soluble IFNAR from urine has an apparent molecular weight of 40-45 kDa (p40). Transcripts for the soluble p40 IFNAR2 are present at the mRNA level and encompass almost the entire extracellular domain of the IFNAR2 subunit with two new amino acids at the carboxy terminal end. There are five potential glycosylation sites on the soluble IFNAR2 receptor. The soluble p40 IFNAR2 has been shown to bind IFN&agr;2 and IFN&bgr; and to inhibit in vitro the anti-viral activity of a mixture of IFN&agr; species (“leukocyte IFN”) and individual Type I IFNs (Novick et al, 1995). A recombinant IFNAR2 subunit Ig fusion protein was shown to inhibit the binding of a variety of Type I IFN species (IFN&agr;A, IFN&agr;B, IFN&agr;D, IFN&bgr;, IFN&agr; Con1 and IFN&ohgr;) to Daudi cells and &agr;/&bgr;
S
subunit double transfected COS cells.
Type I IFN signaling pathways have recently been identified (Platanias et al, 1996; Yan et al, 1996; Qureshi et al, 1996; Duncan et al, 1996; Sharf et al, 1995; Yang et al, 1996). Initial events leading to signaling are thought to occur by the binding of IFN&agr;/&bgr;/&ohgr; to the IFNAR2 subunit, followed by the IFNAR1 subunit associating to form an IFNAR1/2 complex (Platanias et al, 1994). The binding of IFN&agr;/&bgr;/&ohgr; to the IFNAR1/2 complex results in the activation of two Janus kinases (Jak1 and Tyk2) which are believed to phosphorylate specific tyrosines on the IFNAR1 and IFNAR2 subunits. Once these subunits are phosphorylated, STAT molecules (STAT 1, 2 and 3) are phosphorylated, which results in dimerization of STAT transcription complexes followed by n

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