Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Monoclonal antibody or fragment thereof
Reexamination Certificate
1998-06-29
2001-01-23
Mertz, Prema (Department: 1647)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Monoclonal antibody or fragment thereof
C424S130100, C424S139100, C424S141100, C424S142100, C424S143100, C435S326000, C435S334000, C530S388100, C530S388220
Reexamination Certificate
active
06177078
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to monoclonal antibodies (MoAb) and reagents derived therefrom which are reactive with Haemopoietic Growth Factors, and especially with human interleukin 3 (IL-3) receptor &agr;-chain and function as an antagonist to the Haemopoietic Growth Factors.
BACKGROUND OF THE INVENTION
Human interleukin-3 is a pleiotropic cytokine that stimulates the production of hemopoietic cells from multiple lineages including neutrophils, eosinophils, monocytes, megakaryocytes, erythroid cells, basophils and B cells. Recently IL-3 has also been shown to regulate vascular endothelial cell functions; enhancing adhesion molecule expression, neutrophil transmigration and cytokine production. Although some of the effects of IL-3 may be desirable and have prompted its clinical use in bone marrow reconstitution following chemotherapy, it is also apparent that abnormal or excessive production of IL-3 has the potential to lead to disease states. For example, some acute myeloid leukaemias proliferate in response to IL-3, and cells from follicular B cell lymphomas produce and depend on IL-3 for their growth. IL-3 has also been implicated in allergy not only for its ability to stimulate eosinophil and basophil production but also for being a strong stimulus of histamine release from basophils in vitro. The detection of elevated amounts of IL-3 mRNA in the skin and bronchi of allergic individuals further suggests an in vivo role in allergy.
The biological activities of human IL-3 are initiated by the binding of IL-3 to its receptor. This consists of two subunits; an &agr; chain (IL-3R&agr;) which binds IL-3 specifically and with low affinity,
16
and a &bgr; chain (&bgr;
c
) which does not bind ligand on its own but confers high affinity binding when co-expressed with IL-3R&agr;.
17,18
Both chains are required for signalling,
18
however, receptor activation and cellular signalling are dependent on IL-3 binding to IL-3R&agr; as the initial step. The subsequent events are not fully understood but probably involve receptor dimerization leading to the activation of specific kinases associated with the receptor.
19,43
The structure of the extracellular domain of human IL-3R&agr; has not yet been elucidated. Since IL-3R&agr; belongs to the cytokine receptor family, it is predicted to contain a cytokine receptor module (CRM) with two discrete folding domains.
20
In addition there is also an N-terminal domain which, interestingly, has sequence similarities with the human GM-CSF and IL-5 receptor &agr; chains.
21
This feature distinguishes these receptors from the other members of the cytokine receptor family. The functions of the CRM and N-terminal domain of the IL-3R&agr; chain are not known, nor is it known where the IL-3 binding regions lie in the receptor.
SUMMARY OF THE INVENTION
The present invention arises in part as a result of the preparation and the characterization of a monoclonal antibody, MoAb 7G3, directed against the IL-3R&agr; chain which is capable of inhibiting IL-3 binding, IL-3 receptor dimerization
43
and of antagonizing IL-3 functions. These results offer the potential to block IL-3 activity in vivo. A single MoAb to hIL-3R &agr;-chain has been previously described
35
, which recognizes subpopulations of peripheral blood and bone marrow cells. However this antibody does not act as an antagonist to IL-3.
IL-3 is believed to play important roles in both haemopoiesis and inflammation. Although IL-3 has been shown to stimulate several cell types in vitro
37
it is somewhat puzzling that this cytokine has not been detected in the bone marrow or serum of normal animals;
38
suggesting that it is not required for basal haemopoiesis. On the other hand, the injection of IL-3 to mice and humans stimulate haemopoiesis as well as significant side-effects such as bone marrow fibrosis.
39,40
In this respect IL-3 may be viewed as a “reactive” rather than a “steady-state” cytokine and its production may lead to desirable as well as potentially deleterious effects. Consistent with this role, the production of IL-3 is under tight regulatory control in T cells.
41
We show here that MoAb 7G3 is an effective antagonist of IL-3 activities with an ED
50
of 0.4 to 1 nM, consistent with the its K
D
value (FIG.
4
A). Three types of IL-3 functions were studied as the antagonism of IL-3 in these situations is likely to be of clinical significance. Firstly, MoAb 7G3 antagonized IL-3-mediated enhancement of histamine release from basophils (FIG.
6
). Antagonizing IL-3 may be useful in allergic situations as elevated IL-3 mRNA has been noted in the skin and bronchi of atopic individuals,
15
and the presence of IL-3 may lead to excessive stimulation of basophils and eosinophils at allergic reaction sites. Secondly, the IL-3 mediated proliferation of the leukaemic cell line TF-1 was completely antagonized by MoAb 7G3 (
FIG. 5
) at similar concentrations to those described above. Antagonism of IL-3-mediated cell proliferation is likely to be useful in some leukaemias where IL-3 has been shown to promote growth.
10,11
In particular follicular B cell lymphomas which bind IL-3 with high affinity and proliferate in an IL-3-dependent manner
12
may be ideally suited for intervention with MoAb 7G3. Lastly, we found that MoAb 7G3 antagonized IL-3-mediated functions on HUVEC, namely the enhancement of TNF-&agr; stimulation and the synergism with interferon &ggr; (FIG.
7
). The presence of IL-3 receptors on HUVEC and their upregulation by TNF-&agr; and interferon &ggr; has recently been noted,
7,8,34
and their stimulation by IL-3 enhances IL-8 and IL-6 production, HLA class II expression,
8
and neutrophil transmigration.
7
Although the full significance of these in vitro findings need to be ascertained, these effects are likely to contribute to a systemic phase of inflammation and may be amenable to control with MoAb 7G3.
In competition experiments we found that MoAb 7G3 and IL-3 reciprocally inhibited each others binding. This suggests that the IL-3 binding site may lie within or adjacent to the epitope recognized by MoAb 7G3.
We identified the N-terminal domain of IL-3R&agr; as a region required for MoAb 7G3 binding based on the positive immunofluorescence and Western blotting results with a chimeric receptor comprising the N-terminal domain of IL-3R&agr; and the CRM
21
of GM-CSFR&agr;. In contrast, MoAb 7G3 failed to bind to a chimeric receptor comprising the N-terminal domain of GM-CSFR&agr; and the CRM of the extracellular region of IL-3R&agr; (FIG.
8
and
9
). This suggests that the N-terminal domain of IL-3R&agr; is necessary for MoAb 7G3 binding. Further truncations in the N-terminus with retention of MoAb 7G3 reactivity suggest that the 19-49 region of the N-terminal domain of IL-3R&agr; forms part of the epitope recognized by MoAb 7G3. In other experiments we have found that truncation of the N-terminal domain of IL-3R&agr; does not abolish the binding of IL-3 although the affinity of this binding is much decreased. These results have implications for defining the binding site for IL-3 and suggest that this may be formed by two non-contiguous regions in the primary structure of IL-3R&agr;, one of which is in the N-terminal domain and is recognized by MoAb 7G3. The existence of a conformational epitope for IL-3 and MoAb 7G3 is further supported by the inability of MoAb 7G3 to bind linear sequences as represented by the overlapping 14-amino acid peptides.
These results offer the potential to block IL-3 activity in vivo and suggest that the N-terminal domain of IL-3R&agr;, may be involved in ligand binding. An analogy may be drawn to suggest that the N-terminal domain of GM-CSFR and IL-5R&agr; chains, may also be involved in ligand binding because this domain is conserved amongst the IL3R&agr;, GM-CSFR&agr; and IL-5R&agr; family.
21
In one broad form the invention might be said to reside in a monoclonal antibody or antibody fragment with a binding specificity to the N-terminal domain of the IL-3 receptor &agr; chain, or of a homologous region of another hae
Coleman Henry D.
Hamud Fozia
Medvet Science Pty Limited
Mertz Prema
Sudol R. Neil
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