Therapeutic agents as cytokine antagonists and agonists

Details Therapeutic agents as cytokine antagonists and agonists Therapeutic agents as cytokine antagonists and agonists

1998-07-29

2001-03-13

Saoud, Christine (Department: 1647)

Drug, bio-affecting and body treating compositions

Immunoglobulin, antiserum, antibody, or antibody fragment,...

Monoclonal antibody or fragment thereof

C424S130100, C424S139100, C514S002600, C514S023000, C435S007100, C530S389100

Reexamination Certificate

active

06200567

ABSTRACT:

FIELD AND BACKGROUND OF THE INVENTION
This invention relates to antagonists and agonists of cytokines, the therapeutic use of such antagonists and agonists, and method of isolation of such antagonists and agonists.
BACKGROUND OF THE INVENTION
Human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukins IL-3 and IL-5 are cytokines involved in hemopoietic cell survival, production and function (reviewed in Lopez et al, 1992). Because of these properties, IL-3 and GM-CSF are currently being used clinically for bone marrow reconstitution following chemotherapy and radiotherapy (Groopman et al, 1987). However, it is also becoming apparent that excessive or aberrant production of GM-CSF, IL-3 and IL-5 can lead to disease states. For example, elevated amounts of GM-CSF have been found in the lungs of allergic individuals (Kato et al, 1992) and in the joints of patients with rheumatoid arthritis (Williamson et al, 1988). Elevated mRNA for IL-3,GM-CSF and IL-5 have been found in the skin of allergic individuals (Kay et al, 1991). GM-CSF can stimulate the proliferation of leukaemic cells (Young et al, 1986), and IL-3 has been shown to be produced by follicular B cell lymphomas in an autocrine fashion resulting in the proliferation of these cells in an IL-3-dependent manner (Clayberger et al, 1991).
It is clear from these clinical situations that antagonising GM-CSF, IL-3 and IL-5 can be of therapeutic value, and depending on the condition in question, antagonising one of these cytokines may be sufficient.
A number of suggestions for antagonists have already been made, for example in specification PCT/AU89/00177 and in specification PCT/AU94/00432 variants of GM-CSF are identified as antagonists to GM-CSF action however there is no indication that these antagonists are effective for more than only GM-CSF action.
However, in other situations the simultaneous antagonism of all three GM-CSF, IL-3 and IL-5 may be desirable or indeed necessary. For example, eosinophils which are believed to be the major cell type involved in allergy can be maintained in numbers and be stimulated by either IL-3,GM-CSF or IL-5 (Lopez et al, 1989). Antagonism of all three cytokines may thus be necessary to inhibit the actions of eosinophils and basophils. Similarly, basophils which are also believed to play an effector role in allergy can be stimulated by either IL-3,GM-CSF or IL-5 (Lopez et al., 1990) Antagonism of GM-CSF, IL-3 and IL-3 may be accomplished by the concomitant administration of specific antagonists for each different cytokine. Though feasible, this approach has the disadvantage of having to administer up to three different proteins which is not only inconvenient but which also increases the risk of immunogenicity and other side-effects.
SUMMARY OF THE INVENTION
One condition which is prevalent that may be exacerbated by elevated levels of these three cytokines is asthma. The roles of GM-CSF, IL-3 and IL-5 in asthma and allergy have been and continue to be extensively studied. Several studies have shown, by in site hybridisation, increased levels of IL-5 mRNA in lung mononuclear cells (Fukuda et al., 1994; Robinson et al., 1992; Marini et al., 1992; Hamid et al., 1991) and in eosinophils (Broide et al., 1992) in asthmatic patients. Immunochemistry has also revealed increased amount of IL-5 protein in these tissues (Ackerman et al., 1994). In allergen-induced late-phase cutaneous reactions in atopic subjects increased mRNA for IL-5, IL-3 and GM-CSF have been noted (Kay etal., 1991).
BRIEF DESCRIPTION OF THE DRAWINGS
Bronchoalveolar lavage (BAL) fluids from symptomatic asthma patients had greater IL-5 levels than patients with asymptomatic asthma (Sur et al., 1995). Furthermore when symptomatic asthma patients were challenged with antigen in one lung segment, significant levels of IL-5 were noted (980 pg/ml) compared with sham challenged segment of the same patient (2.8 pg/ml) (Sur et al, 1995). In another study with similar design IL-5 levels increased from undetectable to 2800 pg/ml in both allergic and non-allergic asthmatics (Zangrilli et al., 1995). A causal relationship between levels of IL-5 and asthma is suggested from studies in which treatment of moderately severe asthmatic patients with corticosteroids for two weeks resulted in a reduction of the number of cells expressing IL-5 mRNA (Bentley et al., 1996). This reduction was correlated with clinical improvement and also with loss of CD3
+
T cells and activated (EG2
+
) eosinophils.
DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION
GM-CSF has also been detected in the lungs of asthmatics. Indeed in one study of sputum cytokines, GM-CSF appeared to have the dominant effect on eosinophil survival (Adachi et al., 1995).
IL-5 also activates eosinophils to express the EG2 epitope. In several studies increased IL-5 levels were associated with EG2
+
eosinophils in the lung (Fukuda et al., 1994; Bentley et al., 1996). Furthermore there is evidence of a tissue-specific activation of eosinophils. In one experiment, activation of eosinophils in the lung was compared to those in the blood of the same patient. Activation was assessed by cell surface expression of CR-3, p150/95, CD67, CD63 and loss of L-selecting. In patients challenged with endobronchial antigen both peripheral and lung eosinophila were seen at 24h, however only lung eosinophils had increased levels of GM-CSF mRNA suggesting a local activation of these cells. In addition, there was evidence of activation in lung-derived eosinophils but not those from blood. This result is compatible with a specific effect of IL-5 on lung eosinophils and their involvement in asthma.
Animal models also suggest a role for IL-5 in asthma. The most significant data exists with an antibody (TRFK-5) against IL-5 in monkey models of asthma (Mauser et al., 1995). Ascaris-sensitive cynomolgus monkeys were challenged with aerosolised
Ascaris sum
extracts. 0.3mg/kg TRFK abolished increased airway reactivity and diminished eosinophil numbers in BAL fluids. Interestingly this inhibition persisted for 3 months (Mauser et al., 1995). Guinea pig studies supported this conclusion (Mauser et al., 1993). Furthermore mice in which the IL-5 gene was genetically ablated not only had no detectable IL-5 and significant reduction in eosinophil numbers but also developed significantly less severe asthma (as manifest by airways hyperreactivity and lung damage) than IL-5 gene positive litter-mates (Foster et al., 1996). A clear example of restoration of airway responsiveness to methacholine was seen after IL-5 deficient mice were given IL-5 expressing-, but not control-, vaccinia virus infections (Foster et al., 1996).
A likely role for GM-CSF in asthma has also been suggested by over expression of GM-CSF in rat lung leading to eosinophilia, macrophage granuloma and fibrotic reaction, a triad also seen in asthma (Xing et al 1996)
Human interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-5 exert their biological effect by binding to specific surface receptors on the surface of cells (Bagley et al, 1995; Elliott et al, 1989; Park et al, 1989; Lopez et al, 1991). The receptors are heterodimers comprising an a chain which is specific for each ligand, and a &bgr; chain (&bgr;
c
) which is shared between the three receptors (Lopez et al, 1992; Kitamura et al, 1991). Whilst each ligand binds to the respective &agr; chain, &bgr;
c
provides high affinity binding and allows signalling (Miyajima et al, 1992 and U.S. Pat. No. 5,112,961 by Hayashida et al)). The inventors in U.S. Pat. No. 5,112,961 suggest that the high affinity receptor is a valuable tool for screening candidate GM-CSF agonists and antagonists.
Because all three of these cytokines act through a common receptor subunit (&bgr;
c
) we previously hypothesised (Bagley et al, 1995, which reference is incorporated herein), it may be possible to simultaneously inhibit the action of GM-CSF, IL-3 and IL-5 with a single compound. There has, however, to date been no means whereby to approach the problem of findi

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