Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2000-11-20
2004-01-27
Low, Christopher S. F. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S013800, C424S085500, C424S184100, C424S450000, C435S325000, C530S324000, C530S326000, C530S334000, C530S344000, C530S351000
Reexamination Certificate
active
06683052
ABSTRACT:
This application is 371 of PCT/FR 99/00259 filed Feb. 5, 1999.
The present invention relates to lipopeptides containing an interferon-&ggr; fragment, as well as to their use in particular as medicinal products in the context of treating or preventing pathologies against which interferon-&ggr; is liable to have activity by means of at least one of its biological effects, or as an immunity adjuvant which can be used in a vaccine composition for stimulating, orienting or re-orienting the balance of the immune response with respect to any antigen, in particular by promoting the establishment of a type-1 immune response relative to a type-2 response with respect to this antigen.
Cytokines, which are cell immunity components, are classified in two groups: type-1 and type-2 cytokines characterized, respectively, by IL-2, IL-12, interferon-gamma (IFN-&ggr;), IL-4 and IL-5. These two groups regulate and control themselves mutually. They are closely linked to the induction and regulation of the immune response.
The expression “type-1 immune response” means the induction of a type-1 cytokine profile.
The expression “type-2 immune response” means the induction of a type-2 cytokine profile.
On account of its pleiotropic activities, IFN-&ggr; plays a fundamental role in establishing the immune balance. Specifically, this cytokine is involved in various immune defence processes, in particular against viruses, bacteria and protozoan parasites. Furthermore, it inhibits type-2 cytokines and promotes the establishment of a type-1 response which is quite often required to combat certain tumours, and to combat viral and parasitic infections.
Consequently, a therapeutic strategy involving this cytokine appears attractive, but comes up against certain difficulties. These difficulties are the consequences of the short lifetime of IFN-&ggr;, imposing repeated injections of large doses, this often being associated with side effects due to the broad spectrum of activity of the molecule.
The production of recombinant human IFN-&ggr; and its use in human therapy also comes up against the instability of the molecule, which is highly dependent upon the state of glycosylation of the molecule (Saraneva et al., 1995), which is very difficult or impossible to obtain with the expression systems used.
IFN-&ggr; is a glycoprotein of 133 to 143 amino acids depending on the species, which is active in the homodimer form. It acts on the target cells by stimulating a transmembrane receptor, doing so while respecting a species barrier. This species specificity is based on the specific recognition between the outer (or extracellular) portion of the IFN-&ggr; receptor and the N-terminal portion of the cytokine.
The association of IFN-&ggr; and its receptor leads to a dimerization of this receptor and regulates the cytoplasmic association of tyrosine Janus kinase 2 (JAK 2) with the alpha chain of the receptor. This results in a trans- and/or an autophosphorylation of JAK 1 and JAK 2. This represents the first steps in the activation cascade of the transduction pathway of the signal associated with the stimulation by this cytokine, resulting in biological activities such as the induction of the expression of class II CMH molecules, Fc-&ggr; receptors, and adhesion molecules such as VCAM-1.
However, a biological activity independent of the species barrier is observed when the cytokine is delivered inside target cells (in liposomes (Fidler et al., 1985)), or introduced by micro-injection (Smith et al., 1990), or transfection (Sanceau et al., 1987). These results appeared to suggest the existence of an intracellular activation pathway.
This second activation pathway quite probably takes place after interaction of the C-terminal portion of IFN-&ggr; (sequence 95-133 in the case of murine IFN-&ggr;), with the alpha chain of the IFN-&ggr; receptor. This sequence has a binding site which is independent of the species, located at the level of residues 253-287 of the alpha chain of the murine IFN-&ggr; receptor which is of strong affinity (Szente and Johnson, 1994). This intracytoplasmic binding site is located close to the cell membrane and to the JAK 2 tyrosine kinase binding site. This second recognition step appears to be essential to the biological function of this cytokine: the interaction of the C-terminal peptides of IFN-&ggr; with the cytoplasmic portion of the IFN-&ggr; receptor increases the binding of JAK 2 with the alpha chain of the receptor (Szente et al., 1995), which results in an activation of the signal transduction pathway.
These observations supported and explained previous observations regarding the capacity to activate the IFN-&ggr; receptor of murine macrophages with vectorized human IFN-&ggr;. The probable physiological mechanism of action of IFN-&ggr; is thus thought to involve an internalization of the complex formed between IFN-&ggr; and its receptor, following the first recognition step.
Another mechanism may be envisaged, which is thought to correspond to an intracrine stimulation, in the course of which the IFN-&ggr;-producing cells would be autostimulated by the IFN-&ggr; produced inside the cell, without an autocrine activity being necessary via the extracellular portion of the cytokine receptor.
In vitro, the treatment of phagocytic cells of the murine P388D1 cell line (macrophage monocytes) with murine (sequence 95-133) or human (sequence 95-134) IFN-&ggr; peptides for 24 hours at high concentration (100 &mgr;M) can induce expression of the class II CMH receptor. The high concentration required can be explained by the low degree of penetration of the peptide across the cell membrane (by means of pinocytosis activity of the cell studied), or by an inadequate conformation of the peptide, or by a combination of the two phenomena (Szente.et al., 1994).
More recently, Szente identified the structural elements involved in the agonist activity of the C-terminal peptide, and observed that an &agr; helix comprising the unit RKRKR is essential for binding to the cytoplasmic domain of the IFN-&ggr; receptor and for inducing the biological activity (Szente et al., 1996).
Only phagocytic cells have a relative sensitivity to the C-terminal peptide of IFN-&ggr; as used by Szente. These observations are of fundamental interest since they have made it possible to support the hypothesis of the intracrine activity of this cytokine, but they do not propose a product which can be used as such: the biological activity observed with the unmodified peptide moreover did not have the complete spectrum of activity of IFN-&ggr;. In particular, Szente did not obtain induction of the antiviral activity considered as the signature of the IFN-&ggr; activity, and which is used to assay the activity of production batches of this cytokine, since the cells used to carry out the standard test have no phagocytic activity.
The therapeutic use of peptides corresponding to the C-terminal portion of mammalian IFN-&ggr;, such as the above-mentioned murine peptide 95-133 or human peptide 95-134, would be particularly advantageous since it would allow an induction of the biological activity observed in the case of using the whole IFN-&ggr;, by direct binding of the said peptide to the IFN-&ggr; intracellular receptor, without passing via the intermediate step of recognition of the extracellular receptor of this IFN-&ggr;, while at the same time respecting a physiological mechanism of activation if reference is made to an intracrine activity, and thus capable of exhibiting a complete agonist nature.
Specifically, as has been seen above, whole IFN-&ggr; binds first to an extracellular receptor which is specific to a given species. Next, the IFN-&ggr; appears to be internalized inside the cell, and might react with the intracellular portion of the said receptor, which, as has been mentioned above, does not appear to be specific to a given species.
Consequently, the use of the above-mentioned peptides corresponding to the C-terminal portion of mammalian IFN-&ggr; would make it possible to solve this species-barrier problem, and thus to use a pe
Auriault Claude
Gras-Masse Helene
Guillet Jean Gerard
Loing Estelle
Thiam Kader
Institut National de la Sante et de la Recherche Medicale Inserm
Kam Chih-Min
Low Christopher S. F.
Young & Thompson
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