Optimization of Molecular Characteristics

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Not only must we optimize the expression and yield of purified recombinant proteins, we must ensure that the protein is stable, has a high degree of potency and a low degree of toxicity, and has suitable pharmaceutical properties. Pharmaceutical properties encompass distribution and residence time in target tissues, and frequency of dosing, and may have an impact on efficacy and safety.

Starting with a purified recombinant protein, biochemical and biophysical tools are used to elucidate detailed structure and function at the cellular and molecular level. This process elucidates the structure-activity relationship (SAR). The mechanistic elucidation of the protein with respect to target receptor-ligand interactions and enzyme-substrate interactions has led to the construction of macromolecules with enhanced pharmaceutical properties. Some of these enhancements provide (1) increased tissue and target penetration through size reduction of the macromole-cule, (2) rational design of a minimum-binding domain, optimized for receptor binding and resistance to proteolytic degradation, and (3) suitable pharmaceutical properties through domain modification. Some of the strategies in optimization of molecular characteristics are discussed below with specific examples.

and metabolism many years before recombinant DNA technology was introduced (additional details can be found in Chapter 10). Very early on, an enzyme from papaya (papain) was shown to digest the IgG molecule into three fragments: two identical (Fab) fragments capable of binding to a target antigen, and a slightly larger one (FC domain), which does not exhibit antigen binding [25]. Subjecting IgG to an enzyme abundant in the pineapple (pepsin), on the other hand, results in a single cleavage that produces one Fab2 fragment, consisting of an Fab dimer linked via a disulfide linkage, and an Fc fragment that is similar but not identical to that generated by papain (Figure 4.5). For additional details on IgG and fragments, see Chapter 10.

With the availability of antigen binding domains—Fab and Fab2—that are one-third to two-thirds the size of IgG and more compact, the ability of these molecules to permeate across the endothelial cells lining blood vessels and to distribute into tissue is greatly enhanced. As a result antibody fragments, Fab and Fab2, can bind bacterial or tumor antigens found in cells and tissue and reverse the course of disease progression. While this strategy may provide enhanced tissue penetration, some of the functions contributed by the Fc domain, such as increased plasma residence time or prolonged half-life (up to 30 days),as well as Fc-mediated target cell lysis (via complement or cell-mediated mechanisms) will be lost.

Increased Tissue and Target Penetration by Reducing the Size of a Macromolecule

The simplest form of molecular optimization to enhance pharmaceutical properties is exemplified by the modification of the antibody IgG. Because IgG constitutes a major fraction of immunoglobulin and is one of the major proteins found in plasma,it was studied with regard to pharmaceutical properties including stability, distribution,

Chemical cleavage of IgG monoclonal antibody Fab2 + Fc igGc:::;;;;;^^

papain 2 Fab + Fc

Figure 4.5. Schematic representation of enzyme specific cleavage of immunoglobulin G (IgG) by pepsin and papain. Treatment of IgG with pepsin produces two unique fragments, Fab2 with two antigen binding sites and Fc without binding sites. Treatment of IgG with papain generates two Fab and one Fc fragments.

Alternatively, if the prolonged presence of antibody is not required for successful therapeutic application, the rapid antibody removal offered by Fab fragments could be advantageous.A Fabantibody fragment of the monoclonal antibody, 7E3, designed to bind to the glycoprotein receptor on human platelets and inhibit platelet aggregation,is a good example of the benefit of using small antibody fragments. Inhibition of blood clotting is needed for a limited time only, and prolonged inhibition of platelet aggregation could lead to uncontrolled bleeding.

Rational Design of Minimum Binding Domain Optimized for Receptor Binding and Resistance to Proteolytic Degradation

The overall goal of rational drug design is to draw on the knowledge of how a new molecular entity interacts with its target protein to design a therapeutic protein or peptide with superior binding affinity and pharmacokinetic profile. Molecular optimization has long depended on the chemist to design peptide analogues that are resistant to metabolism while retaining biological activity. Peptides derived in this way can be used as a starting point to further refine the molecular structure. This exercise is known as structure-based design and often leads to new molecular entities that have little resemblance to peptides and are not susceptible to peptidases or proteases. New drug candidates that are compact and not susceptible to proteases can be given orally, unlike most protein and peptide drugs, which require parenteral administration to demonstrate activity.

Somatostatin is an endogenous hormone containing 28 amino acids; it regulates a number of hormones including insulin, glucagons, and growth hormone (Box 4.2). Scientists have known for quite some time that a fully active 14 amino-acid peptide fragment contains the active motif of Phe-Trp-Lys-Thr held together by a disulfide bridge through two nearby cysteine resides.

Amino-acid abbreviations are spelled out in Appendix V. Through a series of structure-activity relationship studies, the bioactive conformation and peptide sequences that produce undesirable biologic responses were identified. Also identified were sequences susceptible to proteolysis, and a working-model compound that eliminated these sequences was proposed (Figure 4.6). This allowed the rational design of optimized somatostatin analogues with desirable biologic characteristics and activity and increased stability.

An optimized cyclic octapeptide, Sando-statin (octreotide acetate), is now clinically used as a more potent inhibitor than the parent somatostatin for suppressing growth hormone, glucagon, and insulin. With the reduction of amino acids from 14 to 8, the molecule became more compact and did not bind to alternate sites that could produce undesirable biologic effects (Figure 4.6). In addition, the introduction of the D isomer of tryptophan further resists proteolysis, thereby prolonging pharmacologic activity. An optimized cyclic hexapeptide in which the disulfide bridge is replaced with covalent bonds has been shown to have better pharmaceutical properties than the endogenous hormone (Box 4.3) [26-28].

A similar strategy has been used to optimize a number of peptide-based compounds with therapeutic potential, including tachykinins, enkephalins, and protease inhibitors. HIV protease is essential for producing mature, infectious virus, and two protease molecules are carried in each mature virion (Figure 4.7). With inhibitors developed specifically for HIV protease, but not human protease, one hopes to halt virus replication. Peptides with HIV protease inhibitor activity have been further refined by means of computer- and structure-based design strategies, leading to the development of new molecular entities that are stable to proteases and compact so that they can be administered orally (Table 4.6). Some protease inhibitors (e.g., ritonavir and

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