Hydrophobically-modified protein compositions and methods

Abstract

Hydrophobically-modified proteins and methods of making them are described. A hydrophobic moiety is attached to a surface amino acid residue of the protein. The hydrophobic moiety can be a lipid or a peptide. Alternatively, the protein can be derivatized by a wide variety of chemical reactions that append a hydrophobic structure to the protein. The preferred protein is of mammalian origin and is selected from the group consisting of Sonic, Indian, and Desert hedgehog. The hydrophobic moiety is used as a convenient tether to which may be attached a vesicle such as a cell membrane, liposome, or micelle.

Description

BACKGROUND OF THE INVENTION [0001] It is known that certain proteins exhibit greater biological activity when attached to other moieties, either by formation of multimeric complexes, where the proteins have an opportunity to act in concert, or through other alterations in the protein's physico-chemical properties, such as the protein's absorption, biodistribution and half life. Thus, one current area of research in biotechnology involves the development of methods to modify the physico-chemical properties of proteins so that they can be administered in smaller amounts, with fewer side effects, by new routes, and with less expense. [0002] For example, the binding affinity of any single protein (such as a ligand for its cognate receptor) may be low. However, cells normally express hundreds to thousands of copies of a particular surface receptor, and many receptor-ligand interactions take place simultaneously. When many surface molecules become involved in binding, the total effective ...

AI Technical Summary

Benefits of technology

[0007] In one aspect of the invention, we have solved the problem of finding a way to conveniently make modified forms of biologically active proteins. Methods of the invention can be used to derive multimeric forms of the proteins and / or can be used to change their physico-chemical properties. We have found that modifying a protein (i.e, adding or appending a hydrophobic moiety to an existing amino acid or substituting a hydrophobic moiety for an amino acid) so as to introduce the hydrophobic moiety onto a protein can increase the protein's biological activity and / or its stability. For example, an N-terminal cysteine can be used as a convenient “target” to attach a hydrophobic moiety (e.g., a lipid) and thereby modify biologically active proteins.

[0023] Another method is a method for modifying a protein (such as an extracellular signaling protein) that has an N-terminal cysteine. This method comprises reacting the N-terminal cysteine with a fatty acid thioester to form an amide, wherein such modification enhances the protein's biological activity.

Problems solved by technology

Thus isolated, the low affinity of a protein for its receptor may become a serious drawback to its effectiveness as a therapeutic to block a particular binding pathway, since it must compete against the high avidity cell-cell interactions.

Many of these methods are not highly specific, i.e., they do not direct the point of coupling to any particular site on the protein.

As a result, conventional coupling agents may attack functional sites or sterically block active sites, rendering the coupled proteins inactive.

Furthermore, the coupled products may be oriented so that the active sites cannot act synergistically, thereby rendering the products no more effective than the monomeric protein alone.

However, protective agents have significant biological activity of their own and they may therefore complicate experiments and adversely affect the therapeutic utility of a formulation.

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