Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Recombinant dna technique included in method of making a...
Reexamination Certificate
1997-07-21
2002-07-23
Saunders, David (Department: 1644)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Recombinant dna technique included in method of making a...
C435S243000, C435S320100, C435S325000, C514S002600, C514S008100, C530S350000, C530S363000, C530S395000, C530S868000
Reexamination Certificate
active
06423512
ABSTRACT:
FIELD
The invention relates to fusion polypeptides. It concerns fusion polypeptides comprising an IgE-binding domain and a human serum albumin (HSA) component and salts thereof. It also concerns polynucleotides and physiologically functional equivalent polypeptides which are intermediates in the preparation of such fusion polypeptides; appropriate recombinant expression vectors therefor, corresponding procaryotic and eucaryotic expression systems, and processes for synthesizing the fusion polypeptides.
BACKGROUND
The interaction between immunoglobulin E (IgE) and its receptors has an established role in the defense against parasitic infections in humans (M. Capron and A. Capron.
Science
264 [1994] 1876-1877). In industrialized countries, however, with improved hygienic conditions, the encounter with parasites is less frequent than the disturbance of the IgE network by over-production of IgE in response to environmental allergens. resulting in allergies and other IgE- or IgE-receptor- mediated disease states.
IgE is the primary antibody involved in initiation of an immediate allergic response and is a major participant in the maintenance of the late phase response. IgE is synthesized in B lymphocytes and exerts its effects after binding to the high affinity receptor for IgE, i.e. Fc&egr;RI, which is found on the surface of allergy effector cells such as mast cells, basophils and eosinophils. IgE also exerts inducer functions through binding to its receptor on antigen presenting cells such as Langerhans cells, B cells and monocytes (G. C. Mudde et al.,
Allergy
50 [1995] 193-199).
An allergic response or condition is manifested when IgE molecules bind to the surface of allergy effector cells via the IgE receptor, Fc&egr;RI, and become cross-linked by allergen, thereby effecting signalling to initiate degranulation of cytoplasmic granules in the cells, with accompanying release of mediators of allergy such as histamine, serotonin, prostaglandins and cytokines, and consequent local tissue edema and influx of inflammatory cells. An alternate means of stimulating allergy and associated conditions is the interaction of the IgE receptor, Fc&egr;RI, with circulating autoantibodies against Fc&egr;RI.
Fc&egr;RI typically exists as a tetramer comprising an &agr;-, a &bgr;- and two &ggr;-chains (i.e. “subunits”), although on monocytes and Langerhans cells, the &bgr;-subunit is absent.
The IgE binding site of Fc&egr;RI has been shown to be contained entirely within its &agr;-subunit (referred to as Fc&egr;RI&agr;) (J. Hakimi et al.,
J. Biol. Chem
. 265 [1990] 22079-22081; U. Blank et al.,
J. Biol Chem
. 266 [1991] 2639-2646). Recombinant “knockout” mice genetically deleted for the entire &agr;-subunit have been found to be unable to mount an allergic response to allergen challenge (D. Dombrowicz et al.,
Cell
75 [1993] 969-976).
Fc&egr;RI&agr; is a heavily glycosylated polypeptide of molecular weight about 60 kD, comprising a hydrophobic transmembrane domain as well as hydrophilic extracellular (“ecto-”) and cytoplasmic domains which are exposed to the outer surface of the cell. The IgE binding capability of Fc&egr;RI&agr; has been been further localized to its extracellular portion (J. Hakimi et al. [11990], supra; Leder et al., U.S. Pat. No. 4,962,035). It is possible to produce a soluble, secretable molecule by excising the transmembrane portion and sequences downstream therefrom (C. Ra et al.,
Int. Immunol
. 5 [1993] 47-54); and the resulting truncation, consisting essentially of the human Fc&egr;RI&agr; extracellular domain, has IgE-binding activity in vitro and in vivo (M. Haak-Frendscho et al.,
Immunol
. 151 [1993] 351-358: amino acid residues 1-204 of Fc&egr;RI&agr; fused to a truncated IgGl H chain C region; C. Ra. et al. [1993] supra: residues 1-172 of Fc&egr;RI&agr; therein, corresponding to residues 26-197 of SEQ. ID. NO. 1 hereof). Structural features of this fragment include two potential disulfide bridges and seven potential glycosylation sites (M. Haak-Frendscho et al. [1993], supra).
Therefore, truncations of Fc&egr;RI&agr; consisting essentially of the extracellular domain can potentially be administered therapeutically to a mammal to bind serum IgE in order to prevent its binding to its high affinity receptor on allergy effector cells, and also for suppressing de novo IgE biosynthesis in human lymphocytes (Y. Yanagihara et al.,
J. Clin. Invest
. 94 [1994] 2162-2165).
However, effective use of an IgE-binding polypeptide such as the extracellular domain of Fc&egr;cRI&agr; for systemic treatment of IgE- or IgE receptor- mediated allergic disorders in mammals has been hindered by its extreme transience in vivo due to rapid clearance from circulating plasma. Considering that IgE- or IgE receptor- mediated diseases account for 10-20% of physician-patient contact, an effective treatment would constitute a significant benefit to patients suffering from such conditions and an important advance in the clinical treatment of IgE- and IgE receptor- mediated disorders such as allergy and allergy-related conditions.
It would therefore be beneficial to obtain an IgE-binding polypeptide having prolonged effective serum life and thus improved clinical utility in the treatment of allergy, and in particular the systemic treatment of atopic dermatitis, atopic asthma and chronic urticaria, as well as improved activity in an efficient, cost-effective manner.
SUMMARY
It has now been found that by fusing an IgE-binding domain to a human serum albumin (HSA) component, fusion polypeptides can be obtained having extended serum half-life relative to the IgE-binding domain alone, without loss of IgE-binding activity, resulting in IgE-binding polypeptides indicated for use in the systemic treatment of allergy and other IgE-mediated disorders. Systemically administered IgE-binding polypeptide will bind to serum IgE as well as to circulating auto-antibodies against the IgE receptor, Fc&egr;RI&agr;, preventing them from binding to cell-bound Fc&egr;RI&agr;, and thus preventing and/or inhibiting an allergic reaction and its associated manifestations.
It has further been found that significant improvements in IgE-binding activity can be obtained by using fusion polypeptides of the invention comprising more than one IgE-binding domain per molecule. For example, a “dimer” molecule of the invention has been found to have significantly increased IgE-binding activity relative to a “monomer” of the invention.
The term “dimer” herein refers to a fusion polypeptide of the invention possessing two IgE-binding domains. The term “monomer” refers to a fusion polypeptide of the invention possessing one IgE binding domain. The monomer or dimer may be constructed of a single or multiple HSA components. For example, a monomeric fusion polypeptide of the invention may comprise an IgE-binding domain fused at either its amino or carboxy terminus to an HSA component. Alternatively, the monomer may comprise an IgE-binding domain fused at both of its termini to HSA components. A dimeric fusion polypeptide according to the invention may comprise, for example, two IgE-binding domains fused via the carboxy terminus of one and the amino terminus of the other to an intervening HSA component. Alternatively, the dimer may comprise, in addition to its two IgE-binding domains, multiple HSA components.
It has further been found that a dimer molecule of the invention possesses unexpectedly favorable activity.
The invention therefore is directed to fusion polypeptides and salts thereof comprising at least one IgE-binding domain fused to at least one HSA component; preferably. to monomeric fusion polypeptides, wherein a single IgE-binding domain is fused to one or more HSA components, or to multimeric fusion polypeptides, wherein two or more IE-binding domains are fused to at least one HSA component; more preferably, dimers possessing two IgE-binding domains and at least one HSA component.
The invention is further directed
Digan Mary Ellen
Gram Hermann
Lake Philip
Ferraro Gregory D.
Furman Diane E.
Novartis AG
Saunders David
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