Drug Targeting Organ-Specific Strategies

292 11 Development of Proteinaceous Drug Targeting Constructs
11.6 Recombinant DNA Approaches
The importance of recombinant DNA techniques for the synthesis of drug targeting constructs
is rapidly increasing. This approach offers, in theory, the possibility of generating all
three components of a drug targeting preparation as outlined in Figure 11.1.A carefully chosen
cloning strategy results in a uniform end-product with optimum positioning of the different
components. To obtain such a fully genetically engineered drug targeting construct, all
three components must be peptides or proteins.With respect to the active drug substance this
is likely to be an exception rather than the rule. Some constructs, such as immunotoxins and
immunocytokines, that do fulfil these requirements have been studied extensively in drug
targeting and will be described in detail.
By using recombinant DNA techniques, modifications in the protein backbone, such as additions,
deletions and alterations of amino acids, are easily achieved. These modifications can
contribute to improved pharmacokinetic properties of the construct.Additions may consist of
the introduction of residues that allow covalent conjugation of drug molecules. Deletions of
amino acids can employed to remove membrane-bound regions of a protein, thereby increasing
its solubility. Single amino acid modifications can be used to minimize antibody responses
and alter the binding specificity and/or the three-dimensional structure of a certain protein.
The final requirement of a recombinant DNA approach to the preparation of a drug delivery
construct, is the ability to produce large amounts of the protein. This can by achieved
by bacterial, fungal, insect and mammalian expression systems.The choice of system depends
on how the expression of the protein is regulated, the required purity and yield of the protein,
and whether the protein is toxic to certain types of producer cells. Furthermore, individual
scientists may prefer a particular type of expressing system, depending on laboratory facilities,
safety considerations and production costs. The possibilities and limitations of different
expression systems will be discussed and general guidelines which need to be taken into
account when choosing an appropriate strategy, will be mentioned briefly. Thereafter, with
the aid of several examples, the development and applications of drug delivery constructs obtained
using recombinant DNA technology will be described.
11.7 Recombinant DNA Expression Systems
11.7.1 Heterologous Gene Expression in Escherichia coli
The Gram-negative bacterium E. coli is probably the most widely used host for heterologous
protein production. An obvious advantage of this system is its simplicity. The genetics are
well characterized, the cells grow fast allowing rapid production and analysis of the expressed
protein, and transformation is simple and requires minimal amounts of DNA.
In E. coli foreign genes are normally cloned using inducible promoters such as the lac promoter
that is regulated by the lac repressor and induced by isopropyl β-D-thiogalactopyranoside
(IPTG). This controls gene expression and prevents loss of the gene in situations
where production of the protein might be toxic to the cells. Stronger synthetic promoters, derived
from the lac system, tac and trc promoters, are commercially available. Other common-