Drug Targeting Organ-Specific Strategies

248 9 Tumour Vasculature Targeting

tTF

tumor endothelial cell

tumor endothelium specific target epitope

Figure 9.4. Schematic representation of the mechanism of action of the coaguligand approach. Cross linking of truncated Tissue Factor to tumour endothelial cells leads to local blood coagulation via the tTF/fVIIa complex. tTF, truncated Tissue Factor; fVIIa, factor VIIa; fX (A), factor X (A).

subcutaneous tumours, twice with BsAb * tTF coaguligand led to 38% complete tumour regressions and 24% partial responses [94]. The attractiveness of the coaguligand approach is the use of a truncated form of TF which is devoid of coagulation-inducing activity as long as it is prevented from complexing with the lipophilic factor X on cell membranes. Upon cross linking of the hydrophilic tTF with the target cell membranes by the BsAb, tTF becomes complexed with factor X. In the presence of factor VII/VIIa, this leads to the induction of blood coagulation (Figure 9.4). It is thought that there is a threshold in the number of tTF cross linked to cell membranes, above which the coagulation cascade is initiated. In theory, this allows tumour endothelium-associated target epitopes to be utilized which are highly but not exclusively, expressed on tumour endothelium. The level of expression on other vascular beds is then too low to trigger coagulation after cross linking of the coaguligand.

Using a similar approach of tumour infarction, mouse solid Hodgkin’s tumours spontaneously expressing endothelial VCAM-1 were significantly retarded in outgrowth [95]. The anti-tumour effect was not as dramatic as seen in the MHC Class II model. Possibly, the number of tTF molecules delivered at the site of the tumour endothelium was not sufficient to create a rapid and more or less generalized pro-coagulant situation throughout the tumour vasculature. Only if coagulation is induced in the majority of vessels, will the number of tumour cells deprived of nutrients be sufficient enough to show strong anti-tumour effects. Furthermore, anti-coagulation activities may be strong enough to counteract the coaguligand effects when the kinetics of coagulation induction are insufficient to imbalance local proand anti-coagulation activities.

The coagulation induction potency of coaguligand formulations are likely to be determined by the following factors: (i) the number of target epitopes on the tumour endothelium that allow BsAb-mediated interaction between tTF and factor X on the target cell membrane; (ii) local anti-coagulation activity which may be regulated in a species-specific manner; and (iii) the kinetics of cross linking of the BsAb and the target epitopes in relation to the kinetics of coagulation induction capacity. The number of MHC Class II and VCAM-1 molecules expressed on the tumour vasculature of the animal models discussed, were high, as were the affinities of the antibodies used. This enabled a significant number of tTF to be

rapidly cross linked to the target cell membrane. For clinically relevant target epitopes and targeting devices, the importance of these characteristics needs to be established.

In addition to the targeting of toxins and coagulation-inducing effector moieties to tumour vasculature, inhibitors of angiogenesis-related signal transduction pathways are candidates for selective targeting to tumour endothelium. Although quite effective in various animal models, recent observations of severe toxicity in clinical studies justifies more selective delivery of these molecules into the pro-angiogenic endothelium. At present, however, no data are available on such strategies.

9.3.5 Other Potential Targets

Of the target epitopes suggested for use in tumour vasculature directed drug targeting strategies (Table 9.1), those discussed above seem to be the most promising for development for clinical application.A few have not been extensively studied for this purpose but may also be interesting candidates, and are therefore discussed below.

Endosialin is a cell surface glycoprotein that was identified in various human tumours including sarcomas, carcinomas and neuroectodermal tumours. It comprises a core polypeptide of about 95 kDa and is highly glycosylated (O-linked oligosaccharides). Its biological function and the importance of its expression on tumour vascular endothelium is not yet understood.This antigen is thought to be located on the luminal surface of tumour endothelial cells which represents an obvious advantage for targeting [96]. Apparently, monoclonal antibody (FB5) reactive to endosialin did not show any detectable binding to the vasculature of normal tissues. Although it was suggested that radiolabelled FB5 was rapidly internalized into endosialin-expressing cells, no follow-up on this was reported [96].

Griffioen et al. [97] investigated the potential of targeting the activation antigen CD44. Their studies showed that endothelial cells from tumour vasculature displayed an increased expression of CD44 as compared to endothelial cells from normal tissue. CD44-targeted immunotoxin produced efficient inhibition of CD44-positive endothelial cells with high specificity. Further pre-clinical studies are currently in progress.

Targeted radioimmunotherapy of pulmonary micrometastases was feasible in mice with an antibody directed against thrombomodulin, expressed selectively and in large amounts on the luminal surfaces of capillaries and small blood vessels in the lungs. The short-lived (t1/2 = 45 min) a-particle emitter 213Bi, conjugated to the antibody was delivered to healthy lung and tumour capillaries, resulting in significant tumour growth reduction and an extended life-span of animals treated at low doses. At higher doses, tumours almost completely regressed. However, animals died of lung fibrosis as a result of concurrent damage to healthy tissue [98].

A breakthrough in the search for novel anti-angiogenic compounds occurred when the hypothesis that a primary tumour, while capable of stimulating angiogenesis for its own blood supply, can produce angiogenesis inhibitors which suppress the outgrowth of distant metastases, was proven to hold true. This hypothesis came from the observation that the removal of primary tumours could lead to the accelerated growth of metastases [99]. To test this hypothesis the Lewis lung carcinoma mouse model was used, in which the primary tumour completely suppressed the growth of its metastases. From the urine of these mice a cleavage frag-

250 9 Tumour Vasculature Targeting

ment of plasminogen, called angiostatin, was purified and found to replace the inhibitory activity of the primary tumour completely [100]. Treatment of tumour-bearing mice with angiostatin almost completely prevented metastasis formation in the lung. In theory, these inhibitor proteins could serve as carrier molecules for drug targeting, provided they specifically bind to tumour vasculature. They could then form the basis for dual targeting strategies, in which the carrier itself exerts an effect in addition to the effect of the attached drug. Depending on the mechanisms of action of both active components, synergistic effects might be expected [101].The target for angiostatin on endothelial cells has recently been discovered to be ATP synthase [102]. Whether this binding site is expressed in tumour vasculature and can be exploited as a target epitope with angiostatin as a carrier molecule, needs to be investigated.

Using a similar strategy endostatin was discovered [103]. Although the exact identity of the binding site for endostatin is not known, Chang et al. demonstrated that endostatin can bind to blood vessels of different calibre in various organs. In breast carcinoma binding of endostatin co-localized with FGF-2, but FGF-2 and heparin did not compete for endostatin binding [104]. The lack of selectivity for tumour vasculature probably excludes this molecule from being used as a carrier molecule in drug targeting strategies.

To summarize, some major steps forward have been made in the development of novel drug targeting approaches aimed at selectively killing tumour endothelial cells.The extensive ‘from the bench to the bed’ experience with tumour cell-targeted immunotoxins [105] has paved the way for further development of these tumour endothelial cell-targeted strategies. In this context it is of primary importance that the handling of clinically relevant target epitopes and their drug targeting ligands by endothelial cells, be established under pathological conditions.

9.4Tumour Vasculature Targeting Potentials: Extrapolation of Animal Studies to the Human Situation

From the above, it is clear that tumour vasculature-directed drug targeting approaches to blocking tumour blood flow can be potent strategies for the therapy of large solid tumours. At present, however, only pre-clinical data are available in this area of research, and no sensible extrapolation from pre-clinical experiments with human or animal tumours can be made from the animal model to the clinical setting. One important difference between human tumours and tumours grown in animals is the level of vascular permeability. Although this parameter can vary significantly between the various animal tumours [106], it is believed that the vasculature of animal tumours is in general more permeable. This may be a result of the fact that the majority of animal tumours grow more rapidly than those developing in humans.Another consequence of this rapid tumour growth, is that the majority of blood vessels in animal tumours are in a pro-angiogenic state. As a result, anti-angiogenic therapy or an- giogenesis-related epitope-targeted therapy will affect a greater proportion of the blood vessels in an animal tumour. In human tumours the vasculature is more heterogeneous. Therefore, the selective targeting of drugs to different epitopes covering a broad range of angio- genesis-related markers seems most appropriate strategy to gain access to the majority of tumour blood vessels.

References 251

Many of the drugs that inhibit endothelial cell proliferation, migration and maturation in the angiogenic process act at the level of cell death induction. The thrombo-embolisms observed in the clinic with several anti-angiogenic compounds may indicate that enhanced endothelial cell apoptosis in humans can lead to enhanced micro-thrombus formation and severe toxicity. This observation is in line with the description of the enhanced coagulation-in- ducing capacity of endothelial cells in vitro, when endothelial apoptosis is triggered [107]. Whether the delivery of anti-angiogenic drugs or coagulation-inducing effector molecules into/at tumour endothelial cells via drug targeting will have a similar detrimental effect in humans needs to be carefully addressed.

9.5 Summary and Future Perspectives

Targeting active agents to tumour vasculature to selectively induce tumour blood flow blockade was shown to be highly effective in inhibiting clinically significant tumour burdens in animals. One of the potential advantages of such a treatment may be the absence of drug resistance, as tumour endothelial cells are considered genetically stable. The next step will be to develop similar strategies for use in the clinic. For this purpose, target epitopes on human tumour endothelium need to be identified and studied for their suitability for such strategies. Although some interesting target candidates have been put forward, proof of concept in the human situation needs to be validated. Furthermore, the choice of the drugs to be selectively delivered at or into the pro-angiogenic endothelium will require extensive research in the coming years.

Some 20 different anti-angiogenic agents are currently in clinical trials. Examples of these are marimastat, AG3340, neovastat, TNP-470, thalidomide, CAI, SU5416 , anti-VEGF antibody, and angiostatin (see NCI homepage). It needs to be established whether these drugs can be considered as candidates for use in future drug targeting strategies. Since tumour therapy aims at the complete eradication of tumour cells, a combination of tumour vasculaturedirected strategies (anti-angiogenic drugs as such, or targeted drugs as discussed in this chapter) together with tumour cell-directed chemoand/or immunotherapy, may provide the way forward in the search for optimal treatment for future clinical application.

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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)

10Phage Display Technology for Target Discovery in Drug Delivery Research

Ricardo Mutuberria, Jan-Willem Arends, Arjan W. Griffioen, Hennie R. Hoogenboom

10.1 Introduction

Phage display technology has revolutionized the search for proteins, peptides, and antibodies that bind to molecular targets.The creation of ligand-displaying libraries in combination with powerful selection methods has opened up a wide range of possibilities not only for the search and generation of new ligands amenable for drug targeting, but also in the field of drug discovery and drug design. Libraries containing billions of such ligands can be displayed on phage and enriched for target-binding clones under rationally designed selection techniques, producing molecules with the desired specificity for a given target. The linkage of genotype and phenotype present within the phage particle allows further manipulation of the ligand encoding genes to achieve the desired targeting entity. Selected ligands can be provided with desired effector functions and employed for therapeutic purposes.

For drug targeting applications, where high affinity ligands are needed which specifically recognize celland/or disease-induced surface molecules and are routed into the desired cellular compartments, this technology may be of great importance. This chapter will address how this technology is being used for drug targeting research and target discovery, through the selection of ligands to known and novel targets, and for the engineering and optimization of phage display selected ligands for therapeutic applications. Finally, an update on the current applications of the technology in drug targeting is presented.

10.2 Phage Display Technology

10.2.1 Introduction to the Technology

Phage display libraries have been used extensively for the selection of peptides, antibody fragments or protein variants binding to structures such as proteins, peptides, carbohydrates, nucleic acids or small molecular weight compounds. The ability to rationally design and construct libraries with large molecular diversity makes possible the identification of novel ligands or variants of known ligands with desired binding specificity and characteristics.The display of ligands on the surface of bacteriophage (Figure 10.1) is accomplished by the cloning of proteinor peptide-encoding DNA into the phage genome by fusion to one of the phage coat proteins, pVI, pIII or pVIII. The coat protein fusion is therefore incorporated into the mature phage resulting in the ligand being displayed on the phage surface, while its genetic

256 10 Phage Display Technology for Target Discovery in Drug Delivery Research

Figure 10.1. Display of a Fab fragment on filamentous phage. Fab fragments can be displayed on phage using phagemid vector pCES1 which expresses the heavy chain fragment containing the variable domain and the first constant domain fused to the coat protein gene III, in combination with separate expression of the partner (light) chain. Bacteria harbouring this vector are infected with helper phage to drive the production of phage particles carrying the Fab fragment as a fusion product with the phage coat protein pIII on the surface, while the immunoglobulin encoding genes reside within the phage genome. Alternatively IPTG induction drives the generation of the soluble Fab fragments on the bacterial periplasm. AMPr, ampicillin resistance; H6, histidine tag for purification purposes; MYC, myc tag for detection purposes; A, amber stop codon (TAG) which allows expression of the soluble antibody fragment in non-suppressor strains; gIII, phage gene III; rbs, ribosomal binding site; S, signal sequence directing the expressed protein to the bacterial periplasm; ColE1 ori, E. coli origin of replication; PlacZ, LacZ promoter.

material resides within the phage genome. When the cloned DNA encodes variants of a certain ligand, a phage display library is created. Phage display was first achieved in 1985 by the expression of a peptide on the surface of bacteriophage M13 [1]. Five years later the first random peptide libraries [2–4], and antibody fragment libraries [5] were constructed. Today a large number of moieties have been successfully displayed on the surface of filamentous phages. These include peptides (reviewed by Cesarini et al.) [6], antibody fragments (reviewed by Hoogenboom) [7], enzymes (reviewed by Soumillon et al.) [8], protein scaffolds (reviewed by Nygren and Uhlen) [9], cDNA libraries, (reviewed by Hufton et al.) [10], protease inhibitors [11], transcription factors [12], cytokines [13], and extracellular domains of receptors [14].

In order to retrieve ligands with the desired specificity, phage display libraries are enriched for target binding clones by subjecting the phage libraries to repetitive rounds of selection. This includes incubation with antigen or ‘biopanning’, washing of non-bound phage, elution and re-infection of selected phages into bacteria (Figure 10.2). Antigen binding phage is generally eluted by low or high pH treatment, which drives the dissociation of the ligand from its target without substantially altering the infectivity of the phage for bacteria. A selected filamentous phage is propagated in bacteria, which secrete multiple copies of the phage displaying a particular insert. Selection is repeated until a population of binding clones is enriched

Figure 10.2. The phage display cycle, DNA encoding for millions of variants of a certain ligand (e.g. peptides, proteins or protein fragments) is batch cloned into the phage genome as part of the phage coat protein pIII (coat proteins pVI and pVIII can also be used for display). From this repertoire, phage carrying specific binding ligands can be isolated by a series of cycles of selections on the antigen, each of which involves binding to antigen, washing unbound phage, elution of bound phage and re-amplification in the bacterial cell.

and eventually individual clones are screened for binding to the target. Any procedure that efficiently separates binding clones from those that do not bind, can be used as a selection approach. This has given rise to a large variety of selection methods (Figure 10.3).

Some phage display vectors allow ligands with desired specificity to be produced both as soluble or phage displayed molecules.Alternatively, selected ligands can be recloned for production of the soluble form, or synthesized in vitro as is commonly done for peptide ligands. Phage displayed moieties often exhibit the same or similar functional characteristics as their native counterparts in solution. Soluble forms of the ligand are commonly used to determine relative specificity or affinity to the target. Genetic manipulation of the selected clones allows further optimization of the selected ligand to meet the requirements necessary for drug targeting purposes.