Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-02-14
2004-04-13
Mertz, Prema (Department: 1646)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S326000, C530S388100, C530S388220
Reexamination Certificate
active
06720155
ABSTRACT:
RELATED APPLICATIONS
This application is a §371 of PCT application No. PCT/AU99/00659, filed Aug. 13, 1999.
FIELD OF THE INVENTION
This invention relates to a method of isolating monoclonal antibody inhibitors and reagents derived therefrom and other inhibitors of cytokine binding including monoclonal antibodies and reagents derived therefrom and small molecules capable of inhibiting binding of GM-CSF, IL-3 and IL-5 to the common beta receptor subunit.
INTRODUCTION
Human interleukin (IL)-5, IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) are cytokines involved in hemopoiesis and inflammation (Metcalf; 1986). All three cytokines stimulate eosinophil production, function and survival (Metcalf; 1986) and therefore have the ability to influence inflammatory diseases such as asthma, atopic dermatitis and allergic rhinitis where the eosinophil plays a major effector role. IL-5, being the eosinophil specific cytokine, has received most of the initial attention with IL-5 mRNA and protein levels noted to be elevated in lung tissue and bronchoalveolar lavage (BAL) fluid from symptomatic asthma patients (Fukuda et al 1994). Correlation between IL-5 levels and allergen challenge and disease activity have also been seen (Sur et al, 1996). It is becoming apparent, however, that not only IL-5 but also GM-CSF and IL-3 play a role in eosinophil production and activation in asthma as there is evidence of both GM-CSF and IL-3 being synthesized at sites of allergic inflammation (Bagley et al, 1997b; Allen et al 1997). It is possible that expression of these cytokines contributes to the total number of infiltrating eosinophils and the degree of eosinophil activation. Alternatively, they may be responsible for different phases of eosinophil infiltration. Recent kinetic data from patients undergoing antigen challenge showed that IL-5 levels increased between days 2-7 post challenge, whilst GM-CSF peaked at day 2, and remained elevated throughout day 16. Furthermore, GM-CSF detection extended beyond the site of allergen challenge.
IL-5, GM-CSF and IL-3 stimulate eosinophils and other normal and cancer cells by binding to cell surface receptors that comprise a ligand-specific &agr; chain and a &bgr; chain which is shared by the three receptors (&bgr;
c
) (Bagley et al 1997a). Binding to each receptor &agr; chain is the initial step in receptor activation, however, engagement of either &agr; chain alone is not sufficient for activation to occur. Recruitment of &bgr;
c
by each ligand: &agr; chain complex follows, a step that has two major functional consequences: firstly, it allows the binding of IL-5, GM-CSF and IL-3 to become essentially irreversible; and secondly, it leads to full receptor activation (Bagley et al 1997a). Since &bgr;
c
is the major signalling component of these receptors its engagement leads to the activation of JAK-2, STAT-5 and other signalling molecules culminating in the full plethora of cellular activities commonly associated with either IL-5, GM-CSF and IL-3 stimulation such as eosinophil adherence, priming for degranulation and cytotoxicity, and prolongation of viability (Bates et al, 1996).
In order to block or antagonize the activity of eosinophil-activating cytokines in vivo three major approaches are being tried. One of them utilizes antibodies to the implicated cytokines. For example, antibodies to IL-5 are being used in an animal model of allergen-induced asthma and have shown to have a relatively long lasting effect in preventing eosinophil influx into the airways and bronchial hyperresponsiveness (Mauser et al, 1995). A second approach relies on IL-5 or GM CSF mutants which can bind to the respective &agr; chains with wild type affinity but which have lost or shown reduced ability to interact with human &bgr;
c
. IL-5 mutants such as E13Q, E13K and E13R, and the human GM-CSF mutant E21R directly antagonize the functional activation of eosinophils by IL-5 or GM-CSF respectively (Tavernier et al 1995; McKinnon et al 1997; Hercus et al 1994b). However, at least in the case of E13K, eosinophil survival is not antagonized and in fact this mutant is able to support eosinophil survival (McKinnon et al 1997). A third approach involves the use of soluble receptor &agr; chains which can sequester circulating cytokines. However, this carries the risk of a cytokine: receptor &agr; chain complex potentially interacting with surface-expressed &bgr;
c
and triggering receptor activation. The common theme amongst these approaches is that they tackle a single receptor system involving either IL-5, GM-CSF or IL-3 leaving the other two eosinophil-acting cytokines unaffected. Although the concomitant administration of IL-5 and GM-CSF antagonists may be considered, this may be clinically impracticable.
An alternative approach to blocking eosinophil-activating cytokines involves targeting the common &bgr; chain of their receptors. Although &bgr;
c
does not directly bind IL-5, GM-CSF or IL-3 alone, it does bind to these cytokines complexed to the appropriate receptor &agr; chain. Lopez et al in WO 97/28190, which is incorporated herein by reference in its entirety, have identified the major binding sites of, &bgr;
c
for the IL-5:IL-5R&agr;, GM-CSF:GM-CSFR&agr; and IL-3:IL-3R&agr; complexes. Significantly, these sites are utilized by all three complexes and comprise the predicted B′-C′ loop and F′-G′ loop in &bgr;
c
. Thus targeting &bgr;
c
is not only desirable but also feasible, with the added potential to allow the simultaneous inhibition of IL-5, GM-CSF and IL-3 action by a single agent. These workers have shown that certain mutants in the B′-C′ and the F′-G′ loop fail to bind IL-5, GM-CSF and IL-3.
SUMMARY OF THE INVENTION
The present invention results from the isolation of a monoclonal antibody (BION-1) raised against the membrane proximal domain (domain 4) of &bgr;
c
which is able to block the production and activation of human eosinophils stimulated by IL-5, GM-CSF or IL-3 and blocks the growth of leukaemic cell lines. This MoAb was able to block the high affinity binding of all three cytokines to eosinophils by binding to residues in the predicted B′-C′ and F′-G′ loops of &bgr;
c
, and prevented receptor dimerization and &bgr;
c
phosphorylation. It was found that raising an antibody capable of blocking the binding of all three cytokines was possible by screening monoclonal antibody-expressing hybridoma cell lines arising from immunising mice with cells expressing only domain 4 of &bgr;
c
and lacking domains 1 to 3 and expressing domain 4 and the transmembrane and cytoplasmic regions.
Additionally this finding is likely to have implications for other members of the cytokine receptor superfamily some of which are shared subunits in a given subfamily (that is they bind several cytokines), and some which are ligand specific and bind to only one cytokine. The receptor a-chains for GM-CSF, IL-3 and IL-5 and &bgr;
c
belong to the rapidly expanding cytokine receptor superfamily. Within this superfamily several sub-families are now emerging that are characterized by the sharing of a communal receptor subunit by multiple ligands: gp130 acts as an affinity converter and signal transducer for IL-6 (Hibi et al., 1990; Taga et al., 1992), IL-11 (Hilton et al., 1994), oncostatin M (Liu et al., 1992), ciliary neurotrophic factor, leukaemia inhibitory factor (LIF) (Ip et al., 1992) and cardiotrophin-1(Pennica et al., 1995); the LIF receptor (LIFR) also binds ciliary neurotrophic factor (Davis et al., 1993), cardiotrophin-1 (Pennica et al., 1995) and oncostatin M in addition to LIF (Gearing et al., 1994); IL-2R &bgr; supports affinity conversion and signalling of IL-2 and IL-15 (Giri et al., 1994); IL-2R &ggr; chain affinity converts IL-2 (Takeshita et al., 1992), IL-4 (Russell et al., 1993), IL-7 (Noguchi et al., 1993), IL-9 (Kimura et al., 1995) and IL-15 (Giri et al., 1994); evidence also suggests that IL-4 and IL-13 share a receptor component (Zurawski et al., 1993) and this subunit has recently be
D'Andrea Richard
Lopez Angel
Coleman Henry D.
Coleman Sudol Sapone P.C.
Mertz Prema
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