Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
2000-07-07
2004-03-30
Saunders, David (Department: 1644)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
C514S002600, C530S402000
Reexamination Certificate
active
06713606
ABSTRACT:
This invention relates to polypeptide derivatives, their use in therapy and methods and intermediates for their production.
Essentially all protein drugs are administered as solutions and function in vivo in the solution phase. In biochemist and pharmacology, however, a large number of control and mediator proteins are associated with or function within or on the plasma membranes of cells. Except for soluble, truncated versions of one class of these molecules. no membrane-associated proteins have been developed as therapeutic agents. There are two main reasons for this situation. Firstly. overexpression of proteins that are retained in the membranes of the producer cells is limited by the the low capacity of membranes for proteins and often by the toxic effects of retention when expression is intrinsically efficient. Secondly, extraction of these proteins from membranes requires detergents or organic solvents, often results in inactivation of the protein. leads to difficulties in achieving the high purity needed for drug use and usually gives a product which is hard to formulate for intravenous administration. In addition. retention of very hydrophobic membrane anchoring elements may cause proteins to associate strongly with lipid-binding proteins in blood when administred intravenously thus preventing access to cell membranes.
Soluble, (truncated versions of membrane-associated proteins overcome the production difficulties associated with full length proteins. However such truncated molecules lack the membrane binding capability and specificity of the full length proteins which properties may be advantageous or even essential to the desired therapeutic activity.
The main classes of interaction of proteins with membranes can be summarised as follows:
1. Direct and specific interactions with phospholipid bead groups or with other hydrophilic regions of complex iipids or indirectly with proteins already inserted in the membrane. The latter may include all the types of intrinsic membrane protein noted below and such interactions are usually with exvacellular domains or Sequence loops of the membrane proteins;
2. Through anchoring by a single hydrophobic tranimembrane helical region near the terminus of the protein. These regions commonly prescnt a hydrophobic face around the entire circumference of the helix cylinder and transfer of this structure to the hydrophilic environment of bulk water is energetically unfavourable.
3. Further anchoring is often pmvided by a shon sequence of generally cationic aminoacids at the cytoplasmic side of the membrane, C-terminal to the transmembrane helix;
4. Through the use of multiple (normally 2-12 and commonly 4,7 and 10) transmembrane regions which are usually predicted to be helical or near-helical. Although these regions are normally hydrophobic overall, they frequently show some amphipathic behaviour—an outer hydrophobic face and an inner more hydrophilic one being identifiable within a helix bundle located in the lipid bilayer;
5. Through postranslationally linked phosphatidyl inositol moeities (GPI-anchors). These are generated by a specific biosynthetic pathway which recognises and removes a specific stretch of C-terminal aminoacids and creates a membrane-associating diacyl glycerol unit linked via a hydrophilic carbohydrate spacer to the polypeptide;
6. In a related process, single fatty acid groups such as myristoyl, palmitoyl or prenyl may be attached postranslationally to one or more sites in a protein (usually at N- or C-termini). Again, amino acids (such as the C-terminal CAAX box in Ras proteins) may be removed.
Artificial membranes are considered to be lipid complexes that mimic the basic properties of the cell membrane, i.e., a lipid vacuole with an aqueous interior and a surface chemistry that resembles the cell membrane. The artificial membrane typically contains phospholipids or mimics thereof and may be unilemellar or bilemellar and the outer surface will contain charged groups similar to the choline groups of the most abundant phospholipid. The prototype artificial membrane is known as a liposome and the technologies for the construction of liposomes including the incorporation of therapeutically useful agents into them is well known to those in the art. Liposomes have been evaluated in a number of disease states and liposomes containing the anti-fungal Amphotericin are commercially available. In addition, proteoliposomes have been described. For example, the use of immunoliposomes encapsulating amphotericin B has been reported to be of benefit in the treatment of experimental fungal infections in animal models (e.g. Hospenthal, D. et al (1989) J. Med. Microbiol. 30 193-197; Dromer, F. et al (1990) Antimicrob. Agents Chemother. 34 2055-2060).
Mimics of natural or artificial membranes are often related in structure and will mimic one or more properties of the membrane. One such example is the provision of an artificial surface having pendant groups which mimic the phospholipid zwitterionic groups which are found on the outside of cell surfaces. For example WO92/06719 (Biocompatibles Limited) discloses natural and synthetic phospholipids which may be coated on an artificial surface, e.g. a device which. in use, will come into contact with protein-containing or biological fluids, to provide improved biocompatibility and haemocompatibility and WO 94/16749 discloses additional zwitterionic groups that may be used to improve biocompatibility in a similar way.
The present invention provides a soluble derivative of a soluble polypeptide, said derivative comprising two or more heterologous membrane binding elements with low membrane affinity covalently associated with the polypeptide which elements are capable of interacting, independently and with thermodynamic additivity, with components of cellular or artificial membranes exposed to extracellular fluids.
By ‘heterologous’ is meant that the elements are not found in the native full length protein from which a soluble protein may be derived.
By ‘soluble polypeptide’ is meant a truncated derivative of a full length protein which lacks its natural membrane binding capability, and/or a polypeptide which has a solubility level in aqueous media of >100 &mgr;g/ml.
By ‘membrane binding element with low membrane affinity’ is meant that the element has only moderate affinity for membranes, that is a dissociation constant greater than 0.1 &mgr;M, preferably 1 &mgr;RM-1 mM. The elements preferably have a size <5 kDa.
The derivative should incorporate sufficient elements with low affinities for membrane components to result in a derivative with a high (preferably 0.01-10 nM dissociation constant) affinity for specific membranes. The elements combine so as to create an overall high affinity for the particular target membrane but the combination lacks such high affinity for other proteins for which single elements may be (low-affinity) ligands.
The elements should be chosen so as to retain useful solubility in pharmaceutial formulation media, preferably >100 &mgr;g/ml. Preferably at least one element is hydrophilic.
The invention thus promotes localisation of a therapeutic protein at cellular membranes and thereby provides one or more of several biologically significant effects with potential therapeutic advantages including:
Potency: If the protein is a receptor and an agonist or antagonist activity is localised on the same surface as the receptor itself, an increase in effective concentration may result from the reduction in the diffusional degrees of freedom.
Pharmacokinetics and dosing frequency: Interaction of a derivatised protein with long-lived cell types or serum proteins would be expected to prolong the plasma residence time of the protein and produce a depot effect through deposition on cell surfaces.
Specificity: Many clinically important pathological processes are associated with specific cell types and tissues (for example the vascular endothelium and the recruitment thereto of neutrophils bearing the sialyl Lexis, antigen to ELAM-1, see below). Hence targeting the modif
Dodd Ian
Mossakowska Danuta Ewa Irena
Smith Richard Anthony Godwin
Adprotech Limited
Heller Ehrman White & McAuliffe
Saunders David
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