Drug – bio-affecting and body treating compositions – Conjugate or complex of monoclonal or polyclonal antibody,...
Reexamination Certificate
1998-08-21
2003-08-26
Saunders, David (Department: 1644)
Drug, bio-affecting and body treating compositions
Conjugate or complex of monoclonal or polyclonal antibody,...
C424S131100, C424S139100, C424S185100, C530S326000, C530S327000, C530S328000, C530S387200, C530S387900
Reexamination Certificate
active
06610297
ABSTRACT:
FIELD
This is a 371 of PCT/EP97/01 013, filed Feb. 28, 1997.
The present invention relates to peptide immunogens, and is directed to inhibition of interactions which would normally cause the triggering of mast cells and basophils induced by cell-bound IgE linked to,an allergen resulting in the release of pharmacologically active mediators as well as the de novo synthesis of cytokines involved in the regulation of allergic and inflammatory reactions. It concerns immunogenic molecules including a moiety of a BSW 17 mimotope peptide and their use.
BACKGROUND
Allergic symptoms are brought about through the release of pharmacologically active mediators, notably histamine, leukotrienes and enzymes, from cells into surrounding tissue and vascular structures. These mediators are normally stored or synthesized de novo in special cells known as mast cells and basophil granulocytes. Mast cells are dispersed throughout animal tissue whilst basophils circulate within the vascular system. These cells synthesize and store mediators within the cell, unless a specialized sequence of events occurs to trigger its release.
The role of immunoglobulin E (IgE) antibodies in mediating allergic reactions is well known. IgE is a complex arrangement of polypeptide chains which, as in other immunoglobulins consists of two light and two heavy chains linked together by disulphide bonds in a “Y” shaped configuration. Each light chain has two domains, one variable (V
L
) domain linked to a domain with a relatively invariant amino acid sequence termed a constant domain (C
L
). Heavy chains, by contrast, have one variable domain (V
H
) and in the case of IgE, four constant domains (C
H
1, C
H
2, C
H
3, C
H
4, also known as C&egr;1, C&egr;2, C&egr;3, C&egr;4). The two “arms” of the antibody are responsible for antigen binding, having regions where the polypeptide structure varies, and are termed Fab′ fragments or F(ab′)2, which respresents two Fab′ arms linked together by disulphide bonds. The “tail” or central axis of the antibody contains a fixed or constant sequence of peptides and is termed the Fc fragment. The Fc fragment contains interactive sites which enable the antibody to communicate with other immune system molecules or cells by binding to their Fc receptors.
Fc receptors are molecules which bind specifically to active molecular sites within immunoglobulin Fc regions. Fc receptors may exist as integral membrane proteins within a cell's outer plasma membrane or may exist as free “soluble” molecules which freely circulate in blood plasma or other body fluids. In the human system, high affinity binding of IgE to the receptor Fc&egr;RI is accomplished by a complex protein-protein interaction involving various parts of the third heavy chain constant region domain (C&egr;3) of IgE, and the membrane-proximal, immunoglobulin-like domain (&agr;2) of the Fc&egr;RI&agr; subunit.
Although residues within the C&egr;3 domain of the IgE heavy chain constant region, and regions belonging to the &agr;2 domain of the Fc&egr;RI&agr; receptor, have been identified which are important for binding, the detailed mechanism of the binding process are still obscure. Experimental evidence has been provided by fluorescence energy transfer measurements as well as X-ray and neutron scattering that human IgE adopts a bent structure which is speculated to contribute to the uniquely high affinity of IgE for Fc&egr;RI (Kd~10
−10
M). Moreover, this bent structure is also postulated to be responsible for the equimolar complex between IgE and cell bound or soluble Fc&egr;RI&agr;, although the IgE molecule would provide identical epitopes on the two C&egr;3 domains for receptor binding. This monovalency is a functional necessity if receptor triggering in the absence of allergen is to be avoided (FIG.
1
). Interactive sites, depending on their function, may already be exposed and therefore able to bind to cellular receptors. Alternatively, they may be hidden until the antibody binds to the antigen, whereupon the antibody may change in structure and subsequently expose other active sites which can then trigger a specific immune activity. Based on data obtained from circular dichroism spectra, a conformational rearrangement affecting C&egr;3 upon receptor binding has been proposed as an explanation for the 1:1 stoichiometry of the Fc&egr;/Fc&egr;RI complex on the cellular surface.
For allergic (immunological) release of histamine within the organism from mast cells and basophils, an IgE molecule must lock onto or attach itself with its Fc portion to the cellular Fc receptor site, thus securing the IgE molecule to the mast cell or basophil. The Fab′ portions of the cell-bound IgE molecules must be cross-linked by a particular compatible antigen (the allergen). When such an interaction occurs, the mast cell or basophil is automatically triggered to release histamine to the local environment, manifesting familiar allergic symptoms. Other biochemical events follow in a late phase reaction, resulting in de novo synthesis and release of cytokines and other mediators [Ravetch, J. V., and J. P. Kinet,
Ann. Rev. Immunol
. 9 (1991) 457-492].
Conventional approaches to allergy treatment have involved systemic therapy with anti-histamines or steroids or attempts to desensitize patients; these approaches are not adressed to the basic IgE-mast cell/basophil interaction. Other approaches have been concerned with the production of polypeptide chains capable of blocking the binding of the IgE antibody to the Fc receptors on the cell surfaces and displacing IgE from binding sites upon which IgE is already bound. Moreover, investigations have been carried out in order to define the nature of a putative “effector” site within the IgE Fc region, which was speculated to provide an immunological signal which triggers mast cells/basophils for mediator release.
Using recombinant IgE fragments as immunogens for the generation of a protective anti-IgE vaccine has also been tried and shown to be effective. The main argument against such a vaccine results from the fear that using large IgE fragments for immunization could initiate not only the production of inhibitory antibodies but also generate crosslinking and thereby anaphylactogenic antibodies in the patients (FIG.
2
).
A strategy to overcome this problem would aim at the identification of the smallest IgE fragment possible, ideally consisting of the receptor binding site only, which is buried within the IgE/Fc&egr;RI complex after binding and therefore no longer accessible for crosslinking by the vaccine-generated immune response. Attempts to reconstruct such a complex molecular entity seem unlikely to be successful in view of the spatial distances of the various C&egr;3 regions involved in IgE/Fc&egr;RI interaction.
SUMMARY OF THE INVENTION
It has now been found that the problems intrinsically linked to the “classical” vaccine approach are overcome by using BSW17 mimotopes for active immunization, either as chemically synthesized peptides coupled to appropriate carriers, or as recombinant fusion constructs (e.g. with ovalbumin, IgG, etc.).
BSW17 is a monoclonal antibody which recognizes a conformational epitope on Fc&egr; with at least part of it residing within C&egr;3. The hybridoma cell-line producing monoclonal antibody BSW17 has been deposited on Dec. 19,/1996 with ECACC Salisbury, Wilthuv, SP4 OJE, United Kingdom under the provisions of the Budapest Treaty on the deposit of microorganisms, under deposit number 96121916. This antibody displays an interesting profile of biological activities, as summarized in FIG.
3
. BSW17 or BSW17-like antibodies circulating within the vascular system protect from allergic reactions by
a) inhibiting the triggering of mast cells and basophils through competitive inhibition of the IgE/IgERI interaction and
b) lowering serum IgE levels through downregulation of IgE synthesis at the B cell level.
BSW17 “mimotope” peptides have now been identified by random peptide phage display library screening, i.e. peptides which mimic at l
Kricek Franz
Stadler Beda
Novartis AG
Saunders David
Tung Mary Beth
Wildman David E.
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