Drug Targeting Organ-Specific Strategies

280 11 Development of Proteinaceous Drug Targeting Constructs
overall binding constant compared to the single recognition domain. Particularly in the case
of low affinity ligands, such as sugar molecules, multivalent interactions may be essential for
sufficiently strong binding to the target receptor to take place. In addition, multivalent binding
can contribute to receptor dimerization, a process often observed in the binding of natural
ligands to their receptors [21]. Dimerization and the subsequent intracellular signalling
between the receptor molecules may be an essential step for the internalization of the receptor
and its bound ligand, and thus for receptor-mediated endocytosis of the targeting construct.
Apart from receptor internalization, intracellular signalling by the carrier may result
in a pharmacological response in the target cells. Such carrier-mediated pharmacological activity
may be of therapeutic value or, inversely, may be counter-productive to the therapeutic
actions of the coupled drug moiety.
11.3.1 Carbohydrate Ligands
Historically, the first proteinaceous carriers containing man-made site-directing ligands were
prepared by partial deglycosylation of natural glycoproteins [22]. For instance, desialylation
of bovine fetuin produces asialofetuin, which contains clustered galactose residues at the end
of its carbohydrate antennas. Asialofetuin, and other similarly prepared asiologlycoproteins,
proved efficient carriers for hepatic delivery since the galactose residues are recognized by
the asialoglycoprotein receptor on hepatocytes [23]. Nevertheless, large scale preparation of
such modified proteins is cumbersome and may lead to heterogeneous and enzyme-contaminated
preparations [24].
Another approach to the preparation of glycoproteins is the derivatization of a non-glycosylated
protein, such as serum albumin, with simple sugar residues such as galactose or mannose.
The synthetic preparation of such neoglycoproteins offers the advantages of a more
predictable composition of the carrier and the possibility of large-scale production [25].
Many methods are available for the attachment of sugar groups to side-chain residues of the
protein, either at aromatic groups of tyrosine and phenylalanine side-chains, or at primary
amino groups of lysine residues [26].
Although neoglycoproteins show high affinity for carbohydrate-recognizing receptors
(lectins), they demonstrate only moderate specificity for a specific receptor. This moderate
specificity relates to the relative simple orientation of the oligosaccharide residues compared
to those of the natural glycoprotein ligands which often display multibranched arrays of different
sugar molecules [27]. Consequently, these glycoprotein ligands show a much higher receptor
specificity.
Many approaches have been undertaken to obtain complex carbohydrate ligands with a
high receptor specificity, either by de novo synthesis or by stripping the endogenous oligosaccharides
from bulk amounts of natural glycoproteins [28]. In addition, carbohydrate mimetics
are being developed in which part of the glycosyl antenna is substituted by other functional
groups. For example, the modification of hydroxyl residues in fucose resulted in an increased
specificity for either Kuppfer cells or for a tumour cell line, depending on the type
and position of the modified hydroxyl function [29].
As an alternative to chemical synthesis, complex glycoproteins can be prepared by molecular
biology techniques [30,31]. Typically, the recombinant protein is expressed in mam-