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268 10 Phage Display Technology for Target Discovery in Drug Delivery Research

A phage display-selected scFv directed against CEA has been designed to have effector functions and tested for its potential in antibody-directed enzyme pro-drug therapy (ADEPT). A biologically active recombinant fusion protein containing anti-CEA scFv and the enzyme pseudomonas carboxypeptidase (CPG2) has been produced as a recombinant protein in E. coli and was shown to localize in human colon tumour xenografts. The tumour targeting properties combined with the biological properties of the enzyme can be exploited to induce tumour-specific pro-drug activation, in cases where a non-toxic pro-drug is converted by the action of the targeted enzyme into a highly cytotoxic drug [97].

Finally, phage display may help to address the issue of immunogenicity, particularly of nonhuman antibodies. Antibody humanization is often achieved by grafting CDR-loops into human antibody fragments. However, humanization often requires the replacement of key residues involved in antigen binding in the framework regions, with corresponding residues from the parent non-human antibody. A phage display method that allows selection of framework mutations which increase the binding of humanized antibodies has been described for the humanization of the anti-vascular endothelial growth factor (VEGF) murine antibody A4.6.1 [98]. The humanized version of this antibody is currently in phase II clinical trials to evaluate the inhibitory effect of this antibody-drug on tumour growth and neovascularization. Furthermore, by guided selection [99], a rodent antibody may be rebuilt into a fully human antibody. In two consecutive rounds of chain shuffling, the rodent antibody genes are replaced by human genes, which will mediate binding to a highly similar if not identical epitope on the antigen, differing only in their chemistry of interaction [100].

10.5Discovery of Novel Therapeutics Using Phage Display Technology

Although phage display technology is becoming a widespread research tool, few ligands generated by this technology have reached clinical trials. From the large array of ligands generated by phage display with possible therapeutic applications, there are five antibodies selected from phage libraries currently undergoing clinical trials as drug candidates. Preliminary data with a fully human anti-tumour necrosis factor alpha (TNFα)-neutralizing monoclonal antibody for the treatment of rheumatoid arthritis, demonstrated that the antibody was safe and effective in early clinical trials [101]. Other phage library-derived antibodies that are undergoing clinical trials include antibodies to interleukin 2 (IL-2) for the treatment of autoimmune and inflammatory disorders, an anti-transforming growth factor beta-1 (TGFβ1) antibody as an anti-fibrotic drug, and an anti-TGFβ2 antibody for the treatment of proliferative vitreoretinopathy and prevention of scarring in the eye following glaucoma therapy. A large series of ligands which may have possible therapeutic application are undergoing further characterization and optimization.

Cancer has become a major target in the application of this technology, and human antibodies against tumour antigens such as CEA, EGP-2 and Mucin-1 (MUC-1) are already available. Also a series of peptides and antibodies directed against angiogenesis-related markers such as basic fibroblast growth factor (bFGF), VEGFs and their receptors, tumour-

associated fibronectin, tenascin isoforms, integrins or metalloproteases, have been selected for their potential application in tumour targeting, inhibition of tumour growth and in other angiogenesis-related diseases (see Chapter 9 for drug targeting strategies aimed at angio- genesis-related molecules).

Identification of cell receptor ligands has generated a series of molecules with excellent receptor specificity and occasionally with desired effector functions. This is the case for peptide antagonists of the human estrogen receptor, or cyclic peptides capable of activating the erythropoietin receptors (EPOR) isolated from phage display libraries. Other ligands with effector functions include thrombin inhibitors, viral inhibitors and even intracellular targets such as a reverse transcriptase inhibitory antibody. Table 10.1 lists a number of ligands, which may in the future have widespread application as therapeutic agents.

Table 10.1. Example of phage display ligands with therapeutic applications.

270 10 Phage Display Technology for Target Discovery in Drug Delivery Research

10.6 Conclusions

Phage display has become the most efficient and effective method developed to date for rapidly identifying peptides, antibodies and other proteins that bind to molecular targets. Some of these ligands have biological effector functions or are candidates for ligand-based drug targeting. Identification of the antigens targeted by phage-displayed ligands has been simplified by this technology, by the use of DNA display libraries and by coupling gene isolation and clone identification with affinity selection, facilitating the search for novel targets and biologically important antigens. Selection for function allows the retrieval of ligands with effector functions for use in the generation of drugs. Selection for cellular internalization has opened a new door for the delivery of macromolecular constructs and coupled drugs into the cytoplasm of mammalian cells.

The physical bond between the selected ligand and its encoding gene allows further manipulation of the ligands to obtain optimal affinity and avidity, size, and valency. Furthermore, the coupling of a drug to the targeting ligands can be readily engineered.

The design features required for an optimally-effective drug and/or targeting agent can be readily obtained by phage display technology. Many features give phage display technology clear advantages over conventional approaches for the generation of reagents for drug targeting purposes. These include diversity in protein type and sequence space of combinatorial phage libraries, the power of filamentous phage-based selection, the possibilities of genetic manipulation to generate more effective ligands and specific effector functions, and the adaptability of the system for the production of therapeutic ligands derived from the libraries.

In the near future phage display technology may have further application in the rapid analysis and comparison of protein profiles of large proteomes, such as human cells and tissue.Arrays of phage-displayed proteins such as antibodies or peptides, which selectively bind to proteins from a complex mixture can be generated. Proteins absorbed by the antibody arrays can then be analysed by mass spectrometry. Such technologies will be especially useful for identifying differences between cell or tissue samples such as healthy versus diseased states, and may lead to the identification of drug targets. Once a protein of interest has been identified, its corresponding antibody or peptide ligand can be retrieved and used to monitor protein expression or modification in a range of cell or tissue samples, and can also be used for cloning the target antigen.

Phage display has become a powerful method for the generation of protein-based binding and biologically active reagents. In the forthcoming years an extensive application of this technique is predicted for the development of drugs and drug targeting entities.

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Drug Targeting Organ-Specific Strategies. Edited by G. Molema, D. K. F. Meijer Copyright © 2001 Wiley-VCH Verlag GmbH ISBNs: 3-527-29989-0 (Hardcover); 3-527-60006-X (Electronic)

11Development of Proteinaceous Drug Targeting Constructs Using Chemical and Recombinant DNA Approaches

Robbert J. Kok, Sigridur A. Ásgeirsdóttir, Willem R. Verweij

11.1 Introduction

Several techniques have been developed to selectively increase the accumulation of drugs in specific organs and tissues. One of these drug targeting techniques is the covalent conjugation of the drug to a macromolecule that accumulates at the target site. For this purpose, proteins as well as various other types of (polymeric) macromolecules can be used as drug carriers. This chapter will focus on the design and preparation of proteinaceous drug targeting structures.

Figure 11.1 shows the functional domains that are present in such a drug targeting structure. The core of the construct is the carrier backbone which, in the case of a proteinaceous construct, consists of a protein. Among other factors, the size of the carrier protein has a major influence on the distribution of the drug–carrier construct within the body. More specific targeting of the construct can be achieved by incorporation of site-directing ligands (homing devices) into the protein. By binding to specific receptors, the homing device is instrumental in delivering the construct to its target site. The homing device can be either a conjugated antennary group, or simply an intrinsic domain of the protein. Finally, an essential part of a drug

carrier protein

biodegradable linkage

Figure 11.1. General structure of a proteinaceous drug targeting construct. Functional domains present in a drug targeting construct are (1) the carrier protein, (2) homing devices or site-directing ligands and

(3) the drug moiety. In order to ensure its activation, the drug is often attached via a biodegradable linkage to the carrier.

276 11 Development of Proteinaceous Drug Targeting Constructs

delivery structure is the active drug moiety. Several types of bioactive compounds can be included in a targeting construct. Small drug molecules and other more specifically toxic molecules (toxins) can be linked covalently to the carrier protein. Since the release of the drug from the carrier can be decisive in its pharmacological activity, various types of linkages have been developed which are degraded specifically at the target site. Some of the more generally applicable approaches will be presented here.

As an alternative to linking drug molecules chemically to a core protein, therapeutically active proteins can be used for the preparation of drug targeting constructs. Classically, the latter type of constructs are prepared by chemical modification of the core protein. More recently, proteinaceous drug targeting constructs have also been prepared by recombinant DNA technology. By reconstructing the characteristics of therapeutic proteins at the DNA level and by genetic fusion of functional domains of different proteins into one construct, several interesting new drug delivery approaches have been initiated. These will be addressed at the end of this chapter.

11.2 The Carrier

Since the scope of this chapter is limited to proteinaceous drug targeting constructs, we will not discuss other types of macromolecular drug carriers, such as liposomes and polymeric drug carriers [1–3]. Often, polymeric drug carriers are preferred over proteinaceous drug carriers for their chemical stability, versatility in coupling reactions, high drug loading capacity and lower immunogenicity [2]. However, some general characteristics of proteins have advantages over those of other macromolecular carriers. Proteins are often biodegradable and, generally, biocompatible. Another important feature of a proteinaceous carrier is that the

Table 11.1. Proteins used in drug targeting constructs.

carrier is an homogenous product. In contrast, polymeric carriers are non-homogeneous by nature. Finally, some proteins have unique characteristics that relate to their complex tertiary or quaternary structures. For instance, the binding affinity of the protein to its natural receptor can be the driving force for the selective targeting of the construct. Other interesting carrier proteins are those that are pharmacologically active themselves. Such intrinsically active proteins can be used as active drug substances, or as carriers for small drug molecules in socalled dual active conjugates [4]. Table 11.1 lists examples of carrier proteins that will be discussed in the following sections of this chapter.

11.2.1 Albumin

An important feature of the carrier protein is the size of the macromolecule. Small proteins with a molecular weight lower than about 60 kDa, are rapidly cleared from the bloodstream by glomerular filtration in the kidney [5]. By choosing a carrier protein with an adequately high molecular weight, renal filtration can be prevented. Being sufficiently large to prevent renal filtration, but at the same time small enough for efficient tissue penetration, the albumin molecule has an ideal molecular weight (70 kDa). For this reason, serum albumins from different origins, such as human (HSA), bovine (BSA) or the albumin type autologous to the species in which the conjugate is being tested, have been the carrier of choice for many drug targeting preparations.Two types of drug–albumin conjugates have been reported. First, simple drug–albumin conjugates which accumulate in the target tissue by passive extravasation have been described for the delivery of various anti-cancer drugs to solid tumours [6]. Due to several factors, such as elevated levels of vascular permeability factors and an impaired lymphatic drainage, tumour blood vessels show an enhanced permeability and retention of macromolecules [7]. Preferably, a low number of drug molecules should be conjugated per albumin molecule, since otherwise the construct may undergo enhanced uptake by scavenger receptors in the liver and spleen [8]. On the other hand, specific uptake by scavenger receptors has been exploited for the delivery of anti-inflammatory drugs to the liver [9–11].

The second type of drug–albumin conjugate comprises those in which the albumin protein functions as a backbone for both conjugated drug molecules and homing devices.This type of structure will selectively accumulate in the target tissue by binding to cell surface receptors, a process called active targeting. The binding of the construct to the target cells is primarily driven by the qualities of the homing device, such as type and spatial orientation of the targeting moiety, rather than by the characteristics of the original carrier backbone. Therefore, similar structures can be prepared using carrier proteins other than albumin.

11.2.2 Low Molecular Weight Proteins

As stated earlier, proteins with a molecular weight lower than that of albumin are able to pass through the glomerular membrane in the kidney. Consequently, such low molecular weight proteins (LMWPs) are rapidly removed from the bloodstream. Following glomerular filtration, LMWPs are reabsorbed in the proximal tubular cells of the kidney. Since this process makes the kidney the major catabolic site for these proteins, they can be used as car-

278 11 Development of Proteinaceous Drug Targeting Constructs

riers for renal drug delivery [12,13].A typical example of a protein that has been used for this targeting purpose is lysozyme (LZM, 14 kDa) [14,15] (see Chapter 5 for a more detailed discussion on the development of renal targeting preparations).

11.2.3 Monoclonal Antibodies

Monoclonal antibodies have been extensively reported on as carriers for targeted drug delivery. Starting in the late 1970s, the production of monoclonal antibodies has now evolved into a routine technique that has yielded many potential carrier molecules. Particularly in the field of cancer therapy, monoclonal antibodies are being used for the delivery of diagnostic and therapeutic agents [16] (see also Chapter 8). The original antibodies were of mouse origin, evoking a human anti-mouse antibody (HAMA) immunological response when administered in humans. The use of new recombinant techniques has enabled the preparation of humanized antibodies, in which the mouse recognition domain has been grafted onto a human antibody structure [17].

Whole IgG antibodies with a molecular weight of 150 kDa, are often unable to penetrate tumour tissue as efficiently as smaller molecules [18]. Therefore, smaller antibody fragments and genetically-engineered antibody derivatives have been investigated as drug carriers (see Figure 11.5). These carrier molecules will be discussed in Section 11.8.1.

11.2.4 Transferrin

Some proteins are excellent carriers for drug targeting since they bind to more or less specific receptors on the target cells. In addition to monoclonal antibodies, which in theory can bind to any kind of receptor, several natural ligands for cell-surface receptors have been explored as carrier proteins. This approach is exemplified by constructs that have been developed for targeting via the transferrin receptor (TfR).The transferrin receptor is expressed on most proliferating cells, as well as in a few non-proliferating tissues, among which is the endothelium of brain capillaries. This distribution pattern has inspired the development of transferrin-based constructs and anti-TfR antibodies as carriers for cancer therapy, as well as for the delivery of compounds across the blood–brain barrier [19,20] (see also Chapter 2). With respect to the latter, the ability of the TfR to undergo transcytosis results in the release of the carrier complex in the brain, rather than in endocytosis by the endothelium of the blood–brain barrier. Once inside the central nervous system, the drug delivery construct can bind to TfR-positive cells, such as brain tumours, which can be regarded as a second step in the delivery process.

As an alternative to targeting brain tumours which express the TfR, the transferrin approach can be used for the delivery of fusion proteins which bind to pharmacological receptors inside the central nervous system. An example of this is the construct consisting of nerve growth factor (NGF) and transferrin described in Section 11.8.2.3. The transferrin moiety in this type of construct will enable it to enter the brain, upon which the drug moiety will act by binding to its receptor. This approach seems especially suitable for compounds that cannot pass the blood–brain barrier, such as peptides and other hydrophilic substances.