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REVIEWS LIGAND-TARGETED THERAPEUTICS IN ANTICANCER THERAPY Theresa M. Allen Cytotoxic chemotherapy or radiotherapy of cancer is limited by serious, sometimes lifethreatening, side effects that arise from toxicities to sensitive normal cells because the therapies are not selective for malignant cells. So how can selectivity be improved? One strategy is to couple the therapeutics to antibodies or other ligands that recognize tumour-associated antigens. This increases the exposure of the malignant cells, and reduces the exposure of normal cells, to the ligand-targeted therapeutics. ANTHRACYCLINE Any of a class of antineoplastic antibiotics, including doxorubicin, daunorubicin and epirubicin. LIPOSOME A spherical particle that is formed by a lipid bilayer that encloses an aqueous compartment. POLYMER A macromolecule that is made up of many monomers that are linked by covalent bonds. Department of Pharmacology, University of Alberta, Edmonton, Alberta, Canada T6G 2H7. e-mail: [email protected] doi:10.1038/nrc903 750 Cancer cells share many common features with the normal host cells from which they derive. Consequently, the high levels of selective toxicity that can be achieved with bacterial or viral chemotherapeutics cannot be achieved with anticancer chemotherapeutics because of the lack of unique molecular targets on cancer cells. Most, if not all, cancer chemotherapeutics that are in common use at present — including doxorubicin, vincristine, cyclophosphamide, topotecan and paclitaxel — owe what little selectivity they have for cancer cells to their higher proliferation rates. This can lead to increased toxicities against normal tissues that also show enhanced proliferative rates, such as the bone marrow, gastrointestinal tract and hair follicles. Side effects that occur as a result of toxicities to normal tissues mean that anticancer chemotherapeutics are often given at suboptimal doses, resulting in the eventual failure of therapy; this is often accompanied by the development of drug resistance and metastatic disease. An example of this is the dose-limiting cardiotoxicity that accompanies ANTHRACYCLINE therapy, which results in upper limits being placed on the maximum drug exposure of these otherwise effective drugs. The selective toxicity of an anticancer drug can be increased by either increasing the dose of the drug that reaches the diseased tissue or by decreasing the dose that reaches normal tissues, but, ideally, both will occur. Several approaches for improving the selective toxicity of anticancer therapeutics are being pursued at present. First, newer drugs are being developed that interfere with pathways that are specifically activated in cancer cells. For example, new drugs could interfere with signal-transduction pathways, downregulate proto-oncogenes that are involved in cancer-cell proliferation or interfere with tumour angiogenesis. With a few exceptions — notably imatinib (Glivec), which targets the BCR–ABL oncogene that causes chronic myelogenous leukaemia, and trastuzumab (Herceptin), which targets the ERBB2 (also known as HER2/neu) receptor and is used to treat breast cancer — it is early days for these molecularly targeted therapies, and they will not be considered further. Second, antibody- or ligand-mediated targeting of anticancer therapeutics is being explored. The basic principle that underlies ligand-targeted therapeutics (LTTs) is that the delivery of antineoplastic drugs to cancer cells — or cancer-associated tissues such as tumour vasculature — can be selectively increased by associating the drugs with molecules that bind to antigens or receptors that are either uniquely expressed or overexpressed on the target cells relative to normal tissues. This allows specific delivery of drugs to the cancer cells. The targeting approach can also be applied to drug carriers (microreservoir systems), such as LIPOSOMES and POLYMERS. With the recent developments in antibody engineering and the approval, or imminent approval, of several anticancer therapeutic antibodies for clinical application, the use of antibodies as targeting moieties to increase the selective toxicity of cancer chemotherapeutic agents will be a rapidly growing area of research and a source for new clinical products1,2. | OCTOBER 2002 | VOLUME 2 www.nature.com/reviews/cancer © 2002 Nature Publishing Group REVIEWS Summary • Ligand-targeted therapeutics (LTTs) are a successful means of improving the selective toxicity of anticancer therapeutics. A radioimmunotherapy, an immunotoxin and an immunoconjugate have received clinical approval and over 100 ligand-targeted therapeutics are currently in clinical trials. • Recent advances in antibody engineering have allowed humanized or fully human antibody fragments to be made, which will reduce problems with immune responses against mouse antibodies. Phage-display techniques allow the selection of new targeting moieties that have high affinity for the selected target. • The choice of targeting ligand can be crucial to the success of targeting applications. Variables that must be considered include the degree of receptor expression; whether the ligand is internalized or not; choice of antibody, antibody fragments or non-antibody ligands; and binding affinity of the ligand. • New approaches to LTTs include the use of crosslinked antibody fragments, bispecific antibodies and fusion proteins that carry both the targeting moiety and the therapeutic moiety in the same molecule. • The principles of LTTs can also be applied to microreservoir systems such as liposomes and polymers. Targeting of microreservoir systems can significantly increase the number of therapeutic molecules that can be delivered per targeting molecule and can allow sustained release of the therapy over time. • More basic research needs to be done to understand how to optimize factors such as drug-release rates and pharmacokinetics and biodistribution, and also to understand the mechanisms behind some of the side effects that are caused by some classes of LTTs. • Important issues that need to be addressed include what are the best ways to test LTTs in the clinic, given that they might have their best responses in an adjuvant setting, and how to resolve non-clinical considerations that surround the complex intellectual-property rights in this field. • The principles of LTTs can also be applied to the targeted delivery of gene medicines such as antisense oligonucleotides. So what determines the choice of targeting ligands and drugs in the development of ligand-targeted anticancer therapeutics, and which are proving successful in the clinic? Choice of targeting ligand Some important considerations govern the choice of the ligand that is used to target the drug to the cancer cells. BYSTANDER EFFECT Cells that are killed because they are in close proximity to the actual target, rather than being targets themselves. PHAGE DISPLAY A technique that is used for displaying a peptide (or protein) on the surface of a bacteriophage that contains the gene(s) encoding the displayed peptide(s). Receptor expression. The targeted antigen or receptor should have a high density on the surface of the target cells. For example, a receptor density of 105 ERBB2 receptors per cell was required for an improved therapeutic effect of anti-ERBB2-targeted liposomal doxorubicin over non-targeted liposomal doxorubicin in a metastatic breast cancer model3. Also, target cells should not show a high degree of heterogeneity in their antigen expression. However, if the targeted treatment leads to toxicity against antigen-negative (that is, non-target) cells by nonspecific mechanisms — sometimes termed the ‘BYSTANDER EFFECT ’ (FIG. 1) — then some degree of antigen heterogeneity might be tolerated. The antigen or receptor should also not be shed or downregulated. Circulating shed antigen will compete with the target cells for binding of the targeted therapeutics, and any complexes that form would be rapidly cleared from the circulation. Internalization. The binding of certain ligands to their receptors can cause receptor-mediated internalization (FIG. 1). But whether this is a desired result of the targeted formulation depends on the particular type of targeted therapy. For therapies such as immunoliposomes, internalization might be necessary for optimal results4; for other therapies such as radiolabelled antibodies, internalization is probably immaterial (although internalization might be advantageous for α-emitting radionuclides because of the very short range of α-particles); and for yet other therapies, such as antibody-directed enzyme–prodrug therapy (ADEPT) (FIG. 1), it is important that internalization does not occur because the enzyme must be present at the cell surface to convert non-active prodrugs into active cytotoxic molecules5. Antibody versus non-antibody ligands. So what type of targeting moiety — monoclonal antibodies (mAbs) or antibody fragments, versus non-antibody ligands — should be chosen? Advantages and disadvantages exist for each. A selection of ligands and antibodies that have been used to target LTTs are given in TABLE 1. Non-antibody ligands are often readily available, inexpensive to manufacture and easy to handle. Their principle downside comes from their relatively nonselective expression. Ligands such as RGD, which targets cellular adhesion molecules, and folate and transferrin, which target growth-factor receptors, can bind to some non-target tissues. Also, ligands that occur in the diet, such as folate, can be found in significant levels in body fluids and the free ligand will compete for binding with the targeted therapy. With recent advantages in antibody engineering and PHAGE-DISPLAY technologies, monoclonal antibodies or antibody fragments can be selected that have a high degree of specificity for the target tissue and a wide range of binding affinities can be achieved1,6. Some antibodies — such as trastuzumab (anti-ERBB2, Herceptin) or rituximab (anti-CD20, Rituxan), which bind to the c-ERBB2 proto-oncogene-internalizing surface receptor or to a non-internalizing B-cell surface antigen, respectively — have intrinsic cytotoxicity by means of their NATURE REVIEWS | C ANCER VOLUME 2 | OCTOBER 2002 | 7 5 1 © 2002 Nature Publishing Group REVIEWS a c b d Internalizing target epitope on cancer cell Non-recognized epitope on bystander cancer cell Non-internalizing target epitope on cancer cell Immunoliposome with entrapped active drug Released active drug Antibody–enzyme conjugate for ADEPT Prodrug Activated drug Immunotoxin Toxin ANTI-IDIOTYPIC ANTIBODY An antibody that binds selectively to a specific antigen determinant on a variable domain of an immunoglobulin molecule. As these antibodies can mimic the original antigen, they might compete with the targeting antibody for binding to its epitope. IMMUNOGLOBULIN Any of the structurally related glycoproteins that function as antibodies. They are divided into five classes (IgG, IgM, IgA, IgD and IgE) on the basis of structure and biological activity. FC DOMAIN The antibody fragment that does not contain antigen-combining sites. The Fc domain mediates complement activation and binds to Fc cell-surface receptors — for example, on macrophages. ANTIBODY-DEPENDENT CELLULAR CYTOTOXICITY The lysis of target cells coated with antibody by means of direct cell–cell contact with effector cells bearing Fc receptors — for example, macrophages. COMPLEMENT-DEPENDENT CYTOTOXICITY The lysis of antibody-coated cells by the complement cascade. Cell–cell contact is not required. 752 Figure 1 | Internalization of LTTs and the ‘bystander effect’. a | Binding of the ligand-targeted therapeutics (LTTs) to their target epitopes will, in the case of some antibodies, promote receptor-mediated internalization of the LTT and, following release of the therapeutic intracellularly, lead to cytotoxicity (for example, immunoliposomes and immunotoxins). b | Binding of LTTs linked to noninternalizing antibodies will result in the LTT remaining attached at the target-cell surface (for example, ADEPT (antibody-directed enzyme–prodrug therapy)). c | All the cancer cells will preferably express the target epitope; however, some of the cancer cells might not. Drug that is released into the tumour interstitial space might be taken up non-selectively by cancer cells that do not express the target epitope; this results in cytotoxicity by the ‘bystander effect’ (for example, immunoliposomes and ADEPT). d | Immunotoxins must be internalized to show cytotoxicity, so no opportunity for a bystander effects exists. ability to interfere with molecules that stimulate cell proliferation and differentiation7,8. An added advantage of using these antibodies for LTTs is the possibility of synergy between the signalling antibodies and the chemotherapeutics, because the cells will be targeted in two distinct ways9,10. However, in spite of recent advances in antibody engineering, they remain expensive and time-consuming to produce, and problems with stability and storage might exist. A major problem has been the immunogenicity of rodent antibodies, but this problem is now being effectively addressed (see below). Immune responses to antibodies. The original hybridoma technology for mAb production — described more than 25 years ago — resulted in antibodies that are murine in origin11. Injection of mouse mAbs into humans results in the production of human anti-mouse antibodies (HAMA immune response) or to immune responses against molecules that are linked to the mAbs. This can lead to increased clearance and decreased accumulation of the LTTs in the tumour and, in some patients, to serious immune reactions12. Recent advances in antibody engineering led to the production of chimeric mAbs, then humanized mAbs and, finally, fully human mAbs1 (FIG. 2). These developments will go a long way towards solving the immunological problems that are associated with mAbs, but it remains to be seen if other problematic immune responses might occur — for example, ANTI-IDIOTYPIC responses against humanized or fully human antibodies13. Whole antibody versus antibody fragments. Most mAbs that are used for LTTs fall into the IgG class of IMMUNOGLOBULINS (IgG , IgG or IgG ). The LTTs with 1 2a 2b clinical approval all consist of whole IgG mAbs (FIG. 2) — primarily humanized or chimeric — but there is a trend towards the use of antibody fragments for targeting drug carriers (FIG. 2). One of the advantages of using whole mAbs is the higher binding avidity that comes from the presence of two binding sites on the molecule. The presence of the FC DOMAIN (FIG. 2) in the whole mAb, however, is a mixed blessing, as it can also lead to the mAb binding to normal tissues through Fc receptors, particularly on macrophages, which leads to high liver and spleen uptake of the LTTs and might increase the immunogenicity of the molecule. However, Fc-receptor binding can lead to ANTIBODY-DEPENDENT CELLULAR CYTOTOXICITY and COMPLEMENT-DEPENDENT CYTOTOXICITY, which might enhance tumour-cell kill. Whole mAbs can also be quite stable during prolonged storage. | OCTOBER 2002 | VOLUME 2 www.nature.com/reviews/cancer © 2002 Nature Publishing Group REVIEWS Table 1 | Some ligands that have been used in LTTs Targeting ligands and antibodies Alternative names (trade name) Target Example of tumour target References RGD Cellular adhesion molecules, such as ανβ3-integrin Vasculature endothelial cells in solid tumours 19 NGR Aminopeptidase N (CD13) Vasculature endothelial cells in solid tumours 100 Folate Folate receptor Cancer cells that overexpress the folate receptor 101,102 Transferrin Transferrin receptor Cancer cells that overexpress the transferrin receptor 103,104 GM-CSF GM-CSF receptor Leukaemic blasts 62 Galactosamine Galactosamine receptors on hepatocytes Hepatoma 92 Non-antibody Antibody Anti-VEGFR 2C3 Vasculature endothelial growthfactor receptor (FLK1) Vasculature endothelial cells in solid tumours 105 Anti-ERBB2 Trastuzumab (Herceptin) ERBB2 receptor Cells that overexpress the ERBB2 receptor, such as in breast and ovarian cancers 7 Anti-CD20 Rituximab (Rituxan), ibritumomab tiuxetan (Zevalin) CD20, a B-cell surface antigen Non-Hodgkin’s lymphoma and other B-cell lymphoproliferative diseases 8 Anti-CD22 Epratuzumab, LL2, RFB4 CD22, a B-cell surface antigen Non-Hodgkin’s lymphoma and other B-cell lymphoproliferative diseases 33,52 Anti-CD19 B4, HD37 CD19, a pan-B-cell surface epitope Non-Hodgkin’s lymphoma and other B-cell lymphoproliferative diseases 49,52 Anti-CD33 Gemtuzumab, ozogamicin (Mylotarg) CD33, a sialo-adhesion molecule, leukocyte differentiation antigen Acute myeloid leukaemia 37,67 Anti-CD33 M195 CD33, a T-cell epitope Acute myeloid leukaemia Anti-CD25 Anti-Tac, LMB2 CD25, α-subunit of the interleukin-2 receptor on activated T cells Hairy-cell leukaemia, Hodgkin’s and other CD25+ lymphoma haematological maliganancies Anti-CD25 Denileukin diftitox (Ontak) Interleukin-2 receptor Cutaneous T-cell lymphoma Anti-HLA-DR10β Lym1 HLA-DR10β subunit Non-Hodgkin’s lymphoma and other B-cell lymphoproliferative diseases Anti-tenascin 81C6 Extracellular-matrix protein overexpressed in many tumours Glial tumours, breast cancer Anti-CEA MN-14, F6, A5B7 CEA Colorectal, small-cell lung and ovarian cancers 28,108 Anti-MUC1 HMFG1, BrE3 MUC1, an aberrantly glycosylated epithelial mucin Breast and bladder cancer 28,109 Anti-TAG72 CC49, B72.3 TAG72, oncofetal antigen tumour-associated glycoprotein-72 Colorectal, ovarian and breast cancer 28,110 37 106 46,47 32 107 CEA, carcinoembyonic antigen; GM-CSF, granulocyte–macrophage colony-stimulating factor; LTTs, ligand-targeted therapeutics; NGR, Asn–Gly–Arg tripeptide; RGD, Arg–Gly–Asp tripeptide; TAG72; oncofetal antigen tumour-associated glycoprotein-72; VEGFR, vascular endothelial growth-factor receptor. MULTIVALENCY The ability of an antibody or array of antibodies to bind to several antigen determinants at the same time, which increases the strength of binding between the antibody and its target. F(ab′)2, Fab′ and scFV (single-chain variable) fragments (FIG. 2), lack the Fc domain and the complement-activating region, and this might reduce their immunogenicity14. F(ab′)2 fragments retain two binding regions that are joined by disulphide bonds and can be quite stable during storage. Under reducing conditions, the disulphide bonds are cleaved to yield two Fab′ fragments — each of these contains a thiol (-SH) group that is very useful for coupling the fragments to LTTs. Fab′ fragments and scFV fragments have only one binding domain, which reduces their binding avidity; however, MULTIVALENCY, and hence avidity, can be restored by attaching several fragments at the surface of carriers such as immunoliposomes, or by engineering bivalent or multivalent fragments6. Whereas the use of scFV fragments is attractive because of their ease of identification (for example, from phage display), their ease of production (for example, from Escherichia coli fermentation) and also for their decreased immunogenicity, these small fragments might be less stable during storage than Fab′ fragments or whole mAbs. Binding affinity and ligand density. An interesting question, which is still unresolved, is the choice between high or low binding affinity of the ligand for its antigen or receptor. When the binding affinity is high, there is some evidence that the LTTs have a decreased penetration of solid tumours because of the NATURE REVIEWS | C ANCER VOLUME 2 | OCTOBER 2002 | 7 5 3 © 2002 Nature Publishing Group REVIEWS Aa Mouse IgG Ab Chimeric IgG Antigenbinding site VL Papain cleavage VH Variable region S S S S Light chain S S Contrast region S S Hinge region CHO Fc domain CHO Heavy chains Ac Humanized IgG S S Ad Human IgG S S S S Da scFv C Fab′ S S S Db Bivalent scFv VH SH B F(ab′)2 A A S Dc Bivalent recombinant scFv A B VL Mouse sequences Human sequences Figure 2 | Antibodies and antibody fragments. Targeting antibodies are normally monoclonal immunoglobulin G (IgG) (Aa) or IgG fragments (B–D). F(ab′)2 (B) or Fab′ (C) fragments can be made by enzymatic cleavage of the whole monoclonal antibody (mAb) (Aa) or by molecular biological techniques — for example, Fab′ (C), scFV (Da), bivalent (Db) or recombinant fragments (Dc). mAbs that are made from the traditional hybridoma technique are murine in origin. Recent developments have led to improved techniques for the production of chimeric, humanized or fully human antibodies or fragments (Ab–d). VH, variable heavy chain; VL, variable light chain. TIGHT JUNCTIONS Zones of contact between two adjacent cells that are impermeable to all but the very smallest molecules. EXTRAVASATION Escape of a particle or macromolecule from the bloodstream into the surrounding tissues. TUMOUR WINDOW MODELS A chamber that is attached to the back of an animal — for example, a mouse — that allows the characteristics of an implanted tumour to be observed. 754 ‘binding-site barrier’: the immunotherapy binds strongly to the first targets encountered but fails to diffuse further into the tumour15,16,17 (FIG. 1). For targets in which most of the cells are readily accessible to the LTTs — for example, tumour vasculature and certain haematological malignancies — high binding affinity might be desirable. For carriers such as immunoliposomes and immunopolymers, the issue of ligand density also needs to be addressed. High ligand densities on the liposomes and polymers might be desirable from the perspective of increased binding to target cells, but might be difficult or expensive to achieve. Also, for cases in which the ligand is an intact antibody, high densities have been associated with increased clearance of the immunoliposomes from circulation, which would probably result in decreased localization of the carrier to the target tissue18. Solid tumour vasculature. The unique properties of tumour vasculature have led to the identification of several molecular targets that might be exploited to deliver cytotoxics to the vasculature, killing the cells that are dependent on the tumour vasculature for their oxygen and nutrients. The reader is referred to recent reviews on vascular targeting for a discussion of these molecular targets and the pros and cons of this approach19,20. However, many tumour-targeted LTTs (as opposed to vascular-targeted LTTs) also take advantage of the unique properties of the tumour vasculature. For example, increased permeability of tumour blood vessels helps the localization of LTTs in higher concentrations in solid tumours than in normal tissues. Tumour vessels have an increased permeability to macromolecules owing to a lack of TIGHT JUNCTIONS between adjacent vasculature endothelial cells21,22. The size of the gaps between the cells that line tumour blood vessels has been estimated to be 100–600 nm (REF. 23) — that is, sufficiently large to allow the EXTRAVASATION of most LTTs from the vessel into the tumour interstitial space. Retention of LTTs in solid tumours is aided by their poor lymphatic drainage. However, other factors can lead to incomplete tumour penetration, which can compromise therapy with LTTs. Poor lymphatic drainage can lead to the build-up of higher osmotic pressures within larger tumours and to an outflow of fluids from the tumours, and this might retard or prevent the distribution of LTTs to some regions of the tumour24. So, focal distribution of LTTs has been observed for tumours growing in WINDOW MODELS, with good penetration occurring in some discrete regions of the tumour, and poor or absent penetration in other regions25. As tumours continue to grow, large necrotic areas occur at the tumour centre, which has poor vascularization; the ability of LTTs to penetrate these areas is severely restricted. Distribution of LTTs to tumour cells. Immunotherapeutics are, for the most part, administered via intravenous injection, and are distributed to target cells by means of the bloodstream. When the target cells are in the vasculature or are readily accessible from the vasculature — for example, malignant B or T cells in some haematological cancers, metastatic cells distributed via the bloodstream, or the cells of the tumour vasculature — targeted therapeutics can rapidly bind to these cells, sometimes within just a few minutes, and long circulation half-lives for the LTTs might not be necessary. For distribution of LTTs to more developed solid tumours, it is important that they circulate for sufficient time to result in significant tumour uptake. Particle size can have significant effects on clearance and tumour penetrability of LTTs. In general, smaller particles have slower clearance rates and higher penetration into solid tumour tissue as long as their size is above the exclusion limit for kidney filtration. Larger particles, such as immunoliposomes (approximately 100–150 nm in diameter), can take 48 hours or longer to reach peak levels in the tumour, and it is important not only that the particles circulate for sufficient time to allow for maximum tumour localization, but also that the particles retain their drug contents during this process26. Choice of drug The principles that underlie the choice of drug are simple. In general, the lower the drug to ligand ratio, the more potent the drug needs to be. For example, if the therapeutic is linked directly to individual antibody molecules, then each antibody will deliver, at most, only a | OCTOBER 2002 | VOLUME 2 www.nature.com/reviews/cancer © 2002 Nature Publishing Group REVIEWS 90 a Immunoconstructs Fab′ IgG S S S S scFv SH Toxin, drug, radioisotope or enzyme b Immunoliposomes Fab′ scFv PEG IgG1 Entrapped drug Phospholipid vesicle c Immunopolymers Polymer, e.g. HPMA Degradable spacer Therapeutic agent Figure 3 | Examples of the main classes of LTT. a | Immunoconstructs are formed by the linking of antibodies, antibody fragments or non-antibody ligands to therapeutic molecules, such as toxins (immunotoxins), radioisotopes (radioimmunotherapy), drugs (immunoconjugates) or enzymes (ADEPT). Drug release, if required (immunotoxins and immunoconjugates), occurs through intracellular degradation of the peptide linker. b | Immunoliposomes are formed by the attachment of multivalent arrays of antibodies, antibody fragments or non-antibody ligands to the liposome surface or, as in the example, to the terminus of hydrophilic polymers, such as polyethylene glycol (PEG), which are grafted at the liposome surface. The liposomes contain up to several million molecules of the therapeutic and release of the therapeutic occurs gradually by diffusion down its concentration gradient. c | Immunopolymers are formed by linking both therapeutic agents and targeting ligands to separate sites on water-soluble, biodegradable polymers, such as hydroxypropylmethacrylamine (HPMA), with the use of appropriate degradable spacers to allow for drug release. ADEPT, antibody-directed enzyme–prodrug therapy; LTT, ligand-targeted therapeutic. LINEAR-ENERGY TRANSFER Measurement of the number of ionizations that radiation causes per unit distance as it traverses the living cell or tissue. MYELOSUPPRESSION Causing toxicity against the cells of the bone marrow. few therapeutic molecules to the target cell. If the drug lacks potency, the therapy will fail either because of lack of efficacy, or because large amounts of antibody would have to be used; this could saturate the binding sites, increase the chance of immunological reactions and be expensive to produce. For drug carriers such as liposomes — for which several thousands of drug molecules can be delivered per targeting molecule — drugs with lower potencies will be less problematic. However, issues of drug loading, drug retention and drug-release rate might limit the choice of drugs that are available for association with drug carriers. Individual drugs that have been delivered by different classes of LTTs are discussed below. Recent developments in LTTs Radioimmunotherapy. In radioimmunotherapy (RAIT), mAbs with selectivity for the target cells or tissues are linked to radionuclides (FIG. 3a) with high 131 LINEAR-ENERGY TRANSFER (LET), such as beta ( iodine and yttrium) or alpha (213bismuth and 211astatine) emitters that can cause DNA strand breaks and other effects, resulting in cell kill. The antibodies themselves might contribute to tumour-cell kill through signalling effects — for example, RAIT with constructs containing anti-CD20, which inhibits cell proliferation by interfering with signal transduction and proto-oncogene expression mediated by the B-cell surface epitope CD20. Because radiation can also destroy normal cells, the targeting molecule must achieve a high target to non-target ratio. In general, beta particles, with a penetration range of millimeters, are suitable for therapy of bulk disease, whereas alpha particles, with a penetration range of a few cell diameters, are suitable for micrometastases or circulating tumour cells 27,28. Various therapeutic radiopharmaceuticals are being explored for the treatment of malignant lesions. Several radiolabelled monoclonal antibodies are in clinical trials as therapeutics (TABLE 2). With the exception of two trials that used antibodies labelled with the alpha emitters 213 bismuth and 211astatine, respectively, all the trials involve antibodies that are labelled with 131iodine, a mixed beta and gamma emitter, or 90yttrium, a pure beta emitter. A complication of the 131iodine-containing products is the need to protect the thyroid from damage, because iodine has a natural affinity for the thyroid. The use of a pure beta emitter such as 90yttrium seemed to give better tumour dosimetry than 131iodine, with a lower risk to health-care workers and patient’s families because they are not exposed to potentially damaging gamma radiation from 131iodine (REF. 29). The use of RAIT frequently requires a pretherapy step in which cold mouse antibody is administered either as a predose or concomitantly, which seems to improve the biodistribution of the labelled antibodies to tumours. MYELOSUPPRESSION is the most common dose-limiting toxicity for RAIT28. The HAMA response, which might be expected in patients receiving mouse antibodies, is diminished in patients with non-Hodgkin’s lymphoma who have had previous chemotherapy30, but previously untreated patients have been reported to have developed a significant HAMA response 31. RAIT seems to be most successful in treating haematopoietic malignancies — a high percentage of durable responses has been achieved even at low radiation doses, and particularly in high-dose therapy combined with autologous bone-marrow or stem-cell transplantation. One product, 90yttrium-ibritumomab tiuxetan (Zevalin), which is directed against anti-CD20, has received clinical approval. Other B-cell epitopes that are receiving attention for use in nonHodgkin’s lymphoma are CD22, an internalizing epitope, and the HLA-DR10 β-subunit (Lym1)32–36. T-cell epitopes include CD33 and Tac/CD25 (REFS 37,38). Radiolabelled antibody therapy in cancer and the use of mAb therapy in lymphoma and acute myeloid leukaemia (AML) have recently been reviewed28,39,40. The response to RAIT in solid tumours has not been as encouraging28, as solid tumours tend to be more radioresistant than haematopoietic malignancies. Several types of cancer have undergone clinical evaluation using NATURE REVIEWS | C ANCER VOLUME 2 | OCTOBER 2002 | 7 5 5 © 2002 Nature Publishing Group REVIEWS Table 2 | Radioimmunotherapeutics in the clinic Therapy (trade name) Indication Ligand 90 Yttrium-ibritumomab tiuxetan (Zevalin) NHL (approved) Mouse anti-CD20 IgG1 90 Yttrium-ibritumomab tiuxetan versus rituximab (Zevalin versus Rituxan) Relapsed or refractory low-grade follicular NHL (Phase III) Mouse and human/mouse chimeric anti-CD20 IgG1 131 NHL (Phase III) Mouse anti-CD20 IgG2a 90 NHL and B-cell lymphoma (Phase I/II) Humanized anti-CD22 31,33 131 Diffuse large B-cell lymphoma (Phase I) Mouse anti-HLA-DR10 β-subunit 30,32 213 Acute myeloid leukaemia (Phase I/II) Humanized anti-CD33 35,37 90 T-cell leukaemia (Phase I/II) Humanized anti-Tac/CD25 Iodine-tositumomab (Bexxar) Yttrium-epratuzumab (hLL2) Iodine-Lym1 (Oncolym) Bismuth-HuM195 Yttrium-daclizumab References 9,111 10,112 29,30,31 National Cancer Institute trials NCI-96-C-01471, NCI-97-C-0110F NHL, non-Hodgkin’s lymphoma. 131 BISPECIFIC ANTIBODIES Hybrid, artificially produced antibodies in which each of two antigen-binding sites is specific for separate antigenic determinants. HAPTEN A small molecule, not antigenic by itself, that reacts with antibodies and elicits the formation of antibodies when conjugated to a larger, antigenic molecule. HYPOTENSION Low blood pressure. OEDEMA The presence of abnormally large amounts of fluid in the intercellular tissue spaces of the body. HYPOALBUMINAEMIA An abnormally low albumin content of the blood. 756 iodine, 90 yttrium or 211astatine radiolabels, and a variety of targets including tenascin, CEA (carcinoembryonic antigen), TAG72 and MUC1 epitopes (TABLE 1). In an effort to increase the specificity of the interaction of RAITs with tumour cells (often expressed as tumour to blood ratios) and reduce systemic radiation, new approaches are being explored. In a recent development, humanized crosslinked divalent Fab′ antibody fragments against CEA (huA5B7) were radiolabelled with 131iodine and used in an imaging study in patients with colorectal cancer41. Higher tumour to blood ratios were obtained compared with a murine F(ab′)2 study, indicating that this construct could be superior for use in RAIT; reduced immunogenicity would be expected. Another approach — the affinity enhancement system (AES)42,43 — exploits the concept of pre-targeting and uses BISPECIFIC ANTIBODIES (bsAbs) made from two monovalent antibodies that bind, respectively, to the tumour antigen and a HAPTEN carrier molecule that can be radiolabelled. The non-radiolabelled bsAb is allowed to bind to the tumour, time is allowed for it to clear from circulation, then a radiolabelled carrier — for example, a hapten chelate — that binds to the other arm of the bsAb, is administered. The AES approach resulted in superior results in a xenograft model of human colorectal cancer compared with conventional RAIT44. The RAIT approach has recently been applied to vascular targeting in experiments in which 211astatine, coupled to the lung blood-vessel-targeting monoclonal antibody 201B, eradicated lung tumour colonies in mice45. Extension of these new approaches to human therapy might improve the outcome of RAIT in solid tumours. Immunotoxins. Immunotoxins (ITs) are composed of internalizing mAbs or other ligands that are linked to extremely potent toxins (FIG. 3b), toxin subunits or ribosome-inactivating proteins that kill cells by inactivating protein synthesis or signal transduction. The toxins are derived from plants, fungi or bacteria and some commonly used toxins are ricin toxin, diptheria toxin or Pseudomonas exotoxin. Because the toxins themselves have widespread binding to many normal tissues, which might override the specific binding of the ligand, for inclusion in ITs they are chemically or genetically altered to reduce normal tissue binding. Other toxin molecules such as gelonin, saporin and pokeweek antiviral protein have also been explored. Because of the potent nature of the toxin, very few molecules of ITs need to be delivered to the target cell to effect cell kill. Also, the cell need not be dividing to be killed. Internalization of the IT following binding is a necessary condition of activity and a bystander effect will not occur on antigen-negative cells. So, the ITs tend to be more useful in the treatment of haematological malignancies, which are characterized by a high percentage of malignant cells that express the target antigen. Several ITs are in clinical trials, and one — denileukin diftitox (Ontak) — which is actually an interleukin (IL)-2–diptheria toxin fusion protein rather than an antibody-based drug, has received clinical approval46,47 (TABLE 3). In general, however, ITs have not shown impressive levels of efficacy. They also result in higher levels of systemic toxicity than other therapies, including flu-like symptoms (fever and malaise), acute infusion-related events (shortness of breath, chest and back pain) and a vascular leak syndrome (VLS) that consists of HYPOTENSION, OEDEMA and HYPOALBUMINAEMIA48. Patients who are treated with ITs also frequently experience transient increases in hepatic transaminase levels, which indicates some localization of the toxin to liver. Clinically significant myelosuppression is not normally observed. Toxicities that are associated with the use of ITs preclude chronic use, and their proper niche might be in the treatment of micrometastases or minimal residual disease. Immunotoxicity against either the antibody (for example, HAMA) or the toxin can occur. Although immune responses against antibodies can be reduced by antibody engineering, the toxins themselves cannot be humanized. | OCTOBER 2002 | VOLUME 2 www.nature.com/reviews/cancer © 2002 Nature Publishing Group REVIEWS Table 3 | Immunotoxins in the clinic Therapy (trade name) Indication Ligand/toxin Denilelukin diftitox (Ontak) Cutaneous T-cell lymphoma Interleukin-2/diptheria toxin fragment fusion protein (approved) References Anti-B4-blocked ricin Non-Hodgkin’s lymphoma Mouse anti-CD19/ricin with the galactose binding site blocked (Phase II) 46 49–51 HD37-dgA Refractory B-cell lymphoma Anti-CD22/dgA chains (Phase I) 48 RFB4-dgA Refractory B-cell lymphoma Anti-CD19/dgA chains (Phase I) 48 RFT5-dgA Hodgkin’s lymphoma Anti-CD25/dgA chains (Phase I) 113 Anti-Tac(Fv)-PE38 (LMB2) CD25+ haematological malignancies Anti-CD25 FV fragment/truncated Pseudomonas exotoxin fusion protein (Phase I) 106 RFB4-dsFv-PE38 (BL22) Hairy-cell leukaemia Disulphide-stabilized anti-CD22 FV fragment/truncated Pseudomonas exotoxin fusion protein 114 DT388GMCSF Acute myeloid leukaemia Granulocyte–macrophage colonystimulating factor/diptheria toxin DT388 62 dgA, deglycosylated ricin A. Anti-B4-blocked ricin has been evaluated in several clinical trials, but the results have been disappointing, possibly due to poor penetration of the IT into lymph nodes49. Disappointing results were also found in a trial of this therapy in a paediatric population50. Significant levels of HAMA and/or anti-ricin (HARA) responses were observed in patients who received this therapy51. ITs that contain deglycosylated ricin A chains (dgA) have been evaluated in clinical trials; this eliminated the hepatotoxicity that was observed with the anti-B4blocked ricin, but HAMA and HARA remained a problem, as did dose-limiting VLS, which is more frequent and more severe in patients who have received prior radiotherapy 48,52. Recent research has concentrated on the identification of amino-acid sequences in the toxin that are responsible for VLS, and in determining if sequences that are involved in inhibiting protein synthesis or causing VLS are mediated by different portions of the dgA molecule53–55. In an effort to reduce HARA levels, polyethylene glycol was linked to ricin. This strategy significantly reduced the immunogenicity of ricin, with no effect on its ability to inhibit protein synthesis, so it might be useful in the design of future ITs56. In an interesting attempt at combination chemotherapy, using IT and RAIT together, mouse xenograft models of human B-lymphoma were treated with 131iodine-HD37 (anti-CD22) and/or RFB4-dgA (anti-CD19). The objective was to obtain a bystander effect with the 131iodine-HD37 to debulk the tumour, followed by RFB4-dgA to eliminate residual tumour cells. The order of administration was crucial to the outcome; surprisingly, when IT was administered before RAIT, the therapy was curative in 40% of mice, but when RAIT was administered before IT, the therapy resulted in 50–100% deaths from toxicity57. These results remain to be explained. New approaches to ITs are being explored to overcome problems of toxicity, immunogenicity and heterogeneity of antigen expression58–60. These approaches include the use of genetic engineering to fuse the translocation and catalytic domains of toxins, such as Pseudomonas exotoxin and diphtheria toxin, to fully human single-chain FVs or the use of phage display to select high affinity, tumour-selective ligands. Use of bivalent constructs can also increase the affinity and potency of the antibody fragment. Other activity centres around the selection of ligands that target tumour vascular endothelium and the targeting of oncogene products or differentiation antigens. The use of bivalent constructs increases the affinity of the antibody and increases its potency. Recently, the techniques of phage display and hot-spot mutagenesis were used to increase the affinity of the RFB4(FV) (anti-CD22) by approximately 15-fold, with significant increases in cytotoxicity 61. In a non-antibody approach, a fusion protein of granulocyte–macrophage colony-stimulating factor (GM-CSF, directed against the GM-CSF receptor on leukaemic blasts) and diptheria toxin (DT)388 resulted in modest responses, and significant elevations in liver enzymes were observed, possibly due to the binding of the toxin to macrophages, stimulating the release of cytokines62. Other promising new constructs — which target new epitopes, improve affinities and use toxins that are modified to reduce non-target toxicities — are being tested in animal models or in vitro (TABLE 4). In the future, we can look forward to favourable clinical-trial results for some of these new constructs. Immunoconjugates. The principle that underlies targeted drug conjugates is similar to that underlying ITs, but a few molecules of standard chemotherapy drugs are linked to the targeting molecules, rather than potent toxins (FIG. 3a). Internalization of the conjugate is important for activity, as is release of the drug from the targeting molecule — the conjugate itself is generally inactive. Most conjugates have a relatively low ratio of drug to antibody, in the region of 3–10 drug molecules/antibody, and attempts to increase this ratio can lead to reductions in the binding activity of the antibody. Various drugs have been linked to antibodies, including methotrexate, the vinca alkaloids and the anthracyclines, using various chemical linkers63. NATURE REVIEWS | C ANCER VOLUME 2 | OCTOBER 2002 | 7 5 7 © 2002 Nature Publishing Group REVIEWS Table 4 | New approaches to immunotoxins Construct Description Tumor model A-dmDT390-bisFv Bivalent single-chain anti-CD3, linked to truncated diptheria toxin T-cell malignancies 115 425.3-PE MAb against EGFR, linked to Pseudomonas exotoxin A Gliomas 116 IL-4 (38-37)-PE38KDEL IL-4 (against the IL-4 receptor), linked to a mutated Pseudomonas exotoxin (PE38KDEL) Head and neck squamous-cell carcinoma 117 DT5F11 Fusion toxin between an scFV against GD2, linked to truncated diptheria toxin Neuroblastoma 118 TH69-PE ScFV directed against CD7 on T cells, linked to truncated Pseudomonas exotoxin A Leukaemic T cells 119 ERB-38 [e23(dsFv)PE38] Disulphide-stabilized FV fragments of anti-ERBB2 and a truncated Psuedomonas exotoxin Gastric-cancer-overexpressing ERBB2 receptors 120 VEGF(121)/rGel Tumour vasculature of melanoma or prostate cancers 121 Fusion protein containing VEGF, linked to gelonin References EGFR, epidermal growth-factor receptor; IL-4, interleukin-4; mAb, monoclonal antibody; scFV, single-chain variable fragment; VEGF, vascular endothelial growth factor. Clinical results have been disappointing — for example, a Phase II clinical trial with BR96–doxorubicin had limited activity in metastatic breast cancer, with significant gastrointestinal toxicity 64; this is probably because of the large amounts of antibody that are needed to deliver therapeutic quantities of drugs, low potencies of the drugs, insufficient delivery of the drugs into the target cells even at cell-saturating concentrations, insufficient drug release from the targeting molecule and lack of activity of the conjugates against antigen-negative cells63. Recent developments include the use of carrier molecules such as dextran, human serum albumin or hydroxypropylmethacrylamine (HPMA) to increase the number of drug molecules that can be linked to the targeting molecule. The use of more-potent drugs is also improving the outlook for antibody–drug conjugates — for example, neocarzinostatin, the macrolide antibiotics geldanamycin and maytansin, and calicheamicin, an antitumor antibiotic that is effective at subpicomolar concentrations. A cytotoxic ribonuclease, onconase, of amphibian origin, has also been linked to an anti-CD22 antibody (LL2) and tested in animal models of nonHodgkin’s lymphoma65. The use of human ribonucleases or other human enzymes might be able to circumvent the immunogenicity of plant and bacterial enzymes. Gemtuzumab ozogamicin (Mylotarg) is the only antibody–drug conjugate to receive clinical approval. It consists of a humanized anti-CD33 that is linked to calicheamicin, and is approved for use in AML37. Significant side effects include a high incidence of myelosuppression and elevated hepatic transaminase levels66. Several clinical trials are ongoing — in combination with chemotherapy in AML patients who have either not been treated previously, or are relapsed and refractory — and the responses of other CD33-positive leukaemias to this therapy are also being examined67. ADEPT. An alternative targeting strategy — antibodydirected enzyme–prodrug therapy (ADEPT) — has been designed in the attempt to overcome some of the 758 problems that are associated with the treatment of solid tumours, including poor penetration, antigen heterogeneity, poor drug potency and inefficient drug release. The strategy relies on a two-step approach to targeting (FIGS 1,3a). In the first step, mAbs are used to localize enzymes to tumour-cell surface antigens. After any unbound antibody–enzyme conjugate has cleared from circulation, prodrugs are administered, which are converted to active drugs by the targeted enzyme. This strategy requires antibodies that are non-internalizing, so that the drug is released outside the cell by enzymes at the cell surface. Passive uptake of the drug into the target cells will then occur, and good opportunities exist for a bystander effect against antigen-negative cells. One of the principle strengths of ADEPT is its potential to generate high concentrations of drug intratumorally. Many enzymes and prodrugs are available for use with the ADEPT technology 5,68. Enzymes include β-lactamase (which cleaves β-lactam rings to release active drugs such as paclitaxel and doxorubicin), cytosine deaminase (which converts 5-fluorocytosine to the active drug 5-fluorouracil) and carboxypeptidase G2 (CPG2; which cleaves terminal glutamic-acid amides to produce nitrogen mustards). In a Phase I clinical trial in patients with advanced colorectal cancer, patients were given CPG2 linked to an anti-CEA F(ab′) 2 (A5B7). This was followed by administration of a clearing agent, galactosylated anti-CPG2, which accelerated the clearance of the antibody–enzyme conjugate from the blood. Finally, a benzoic-acid mustard-glutamate prodrug was administered. All patients developed a significant immune response against the bacterial enzyme, and antitumour responses were modest 5,69. Current research in this area revolves around the use of fusion protein constructs and enzymes that are relatively non-immunogenic in order to address issues that have arisen with regard to immunogenicity and heterogeneity of the mAb–enzyme conjugates 5,69,70. A fusion protein between anti-CEA scFV and CPG2 has been constructed and showed excellent tumour to | OCTOBER 2002 | VOLUME 2 www.nature.com/reviews/cancer © 2002 Nature Publishing Group REVIEWS Box 1 | Passive targeting Clinically approved therapeutics (or new drugs) can be stably associated with sustained-release microreservoirs (drug carriers), such as liposomes or polymers. If the carriers are sufficiently small in diameter (usually less than 150 nm), they have been shown to extravasate and localize to areas of increased vascular permeability, such as those found in angiogenic tumours, in a process that is referred to as passive targeting. This results in significant increases in the amount of drug delivered to solid tumours relative to the free drug. At the same time, peak drug levels and distribution of the drugs to normal tissues is decreased, leading to fewer side effects. A notable clinical example is the significant decrease in cardiotoxicity observed for the liposomal anthracycline drugs. Liposomal formulations of the anthracycline drugs doxorubicin (Doxil, also marketed as Caelyx) and daunorubicin (DaunoXome) have been on the market for several years and another liposomal doxorubicin formulation has recently received clinical approval (Myocet). Many other liposome-associated anticancer drugs and at least one polymer–doxorubicin formulation are in clinical trials. The reader is directed to the literature for discussions of these formulations, which do not fall under the classification of LTTs. normal-tissue ratios in xenograft models of human colon adenocarcinoma71. Further clinical trials are needed to establish the utility of ADEPT for cancer therapy. PHARMACOKINETICS The study of the fate of drugs in the body over a period of time, including the processes of absorption, distribution, localization in tissues (biodistribution), metabolism and excretion. Immunoliposomes. In the LTTs discussed above, radiolabels, toxins or drugs were linked (or fused) directly to an antibody, antibody fragment or other ligand. Immunoliposomes use a different approach: chemotherapeutic molecules or gene therapeutics are loaded into liposomes and targeting ligands are attached at the liposome surface — or preferably to the terminus of polymers such as polyethylene glycol (PEG) that are grafted at the liposome surface — using conventional chemical-coupling techniques72,73 (FIG. 3b). Alternatively, clinically approved liposomal drugs such as Doxil can be converted into targeted formulations by a versatile ‘post-insertion’ technique, which allows preformed, preloaded liposomes to be converted into immunoliposomes that contain the ligand of choice by a simple incubation step74–76. The advantage of the post-insertion approach is that the passive targeting and anticancer activity of a non-targeted liposomal drug can be further improved by converting it, as needed, into a targeted liposomal drug using antibodies or ligands that can be tailored for individual patients or particular cancers. By using approved nontargeted drugs, this can be done without imparting unacceptable levels of manufacturing complexity to the production of individual targeted therapies. One advantage to the immunoliposome approach is the ability to deliver several tens of thousands of molecules of drug with a few tens of ligand molecules per liposome, resulting in extremely high drug to ligand ratios. Another advantage is multivalent presentation of targeting molecules, which can increase the avidity, uptake and signalling properties of antibody fragments. The opportunity also exists for additivity or synergy between signalling antibodies at the liposome surface and the liposomal drug9,10. One potential disadvantage of immunoliposomes is their large size (usually around 100 nm in diameter) relative to other constructs, which can compromise their penetration of solid tumours. Increasing vasculature permeability through the use of chemicals or hyperthermia has been used to increase the localization of liposomes to specific tissues77,78. Problems with the premature removal of immunoliposomes from circulation into macrophages of liver and spleen have largely been overcome with the use of sterically stabilized liposomes and new technologies for linking ligands to the terminus of molecules such as polyethylene glycol, which is anchored in the liposome bilayer 4,75,79–81. Unlike the therapies described above, immunoliposomes function as drug infusions, with sustained release of the drug occurring over periods as long as several days depending on the drug release rates that are engineered into the product. The PHARMACOKINETICS and biodistribution of the carrier-associated drugs are substantially altered relative to the non-associated (free) drugs. The carrier can also protect the drugs from premature degradation, prolonging their activity in vivo, or can be used to solubilize drugs that are difficult to formulate. Until recently, most of the activity in the liposome field has concentrated on passive targeting, rather than ligand-mediated (‘active’) targeting (see BOX 1). Following binding of the liposomes to target cells, delivery of the therapeutic to the cell occurs by one of two mechanisms, depending on whether the ligand is internalizing or non-internalizing (FIG. 1). After liposomes that are linked to a non-internalizing ligand bind to target cells, the drug is gradually released from the liposomes and is taken up by the cell as free drug, using standard uptake mechanisms. When the ligand is an internalizing one, the liposome–drug package is taken into the cell by receptor-mediated endocytosis and, assuming it is stable in the environment of the endosome, the drug is gradually released within the cell. The numbers of drug molecules that are delivered intracellularly are higher when an internalizing ligand is used — diffusion and redistribution of the released drug seem to be higher for non-internalizing antibodies, which leads to lower concentrations of drug being delivered to the target cells. It is probably for this reason that internalizing ligands have resulted in better therapeutic outcomes in animal models82. For internalizing ligands, because not all of the liposome–drug packages will immediately be internalized into target cells, the opportunity for a bystander effect exists, as a drug that is released extracellularly diffuses within the tumour to be taken up by antigen-negative cells. Phage technology is now being used to identify antibodies that are efficiently internalized for use in conjunction with immunoliposomes83. No immunoliposomal preparations have reached the clinic to date. Based on their activities in animal models, prime candidates for progression to clinical trials include liposomal chemotherapeutics that target via anti-ERBB2 (REFS 3,84) and anti-CD19 or other B-cell or T-cell epitopes18,85. An important question concerning the use of immunoliposomes, as yet unresolved, is whether multivalent presentation of a ligand on a carrier increases the immunogenicity of either the targeting ligand or liposome components. Some recent NATURE REVIEWS | C ANCER VOLUME 2 | OCTOBER 2002 | 7 5 9 © 2002 Nature Publishing Group REVIEWS evidence indicates that the antigen density on target cells might be crucial in the therapeutic response to immunoliposomal drugs84. A review of the literature indicates that immunoliposomal drugs have an improved therapeutic outcome when used in an adjuvant or minimal residual-disease setting82. Because liposomal drugs are not biologically active until they are released, studies on how the rate of drug release affects therapeutic outcome and on how to optimize the payout rate of drugs are becoming important areas of research86–88. Difficult to solubilize drugs — for example, hydrophobic drugs such as paclitaxel89 — might present a particular problem for delivery in immunoliposomes as these drugs rapidly partition out of liposomes in vivo, making it unlikely that they will be delivered to the target cell by immunoliposomes. Immunopolymers. Bioconjugates of drugs to biodegradable water-soluble polymers, such as the copolymer HPMA, are beginning to be explored as targeted drug carriers. As with immunoliposomes, immunopolymers can change the pharmacokinetics and biodistribution of the bound drugs and take advantage of the passive targeting phenomenon (described in BOX 1). Release of the drug from the polymer is necessary for its biological activity, so biodegradable spacers have been introduced between the polymer backbone and the drug molecule — for example, the tetrapeptide spacer Gly-Phe-Leu-Gly, which can be cleaved by cathepsin B within the lysosomal compartments of target cells following uptake of the carrier by pinocytosis. It is obvious that polymer–drug conjugates would benefit from ligandmediated targeting using internalizing ligands, given the requirement of metabolic activation for drug release. Random conjugation of mAbs with polymers can cause loss of antibody activity, so a new approach was adopted in which methacrylamide-Fab′ fragments, with a polyethylene glycol spacer, were co-polymerized with HPMA and drug monomers, with a cleavable peptide spacer 90 (FIG. 3c). Various targeting agents have been examined, including Fab′ from mAb OA-3 against ovarian cancer90, lectins90 and triantennary galactose91. A Phase I study of a galactosamine-targeted HPMA polymeric doxorubicin, with a Gly-Phe-Leu-Gly spacer, in hepatoma patients showed some evidence of clinical response and clear evidence of liver targeting92. An interesting recent study has compared the intracellular fate of free doxorubicin with that of antiThy1.2-targeted or anti-CD71-targeted HPMA-doxorubicin. In contrast to free drug, no drug from the targeted conjugates could be detected in the nuclei of the target cells for up to 72 hours of incubation, and no evidence of apoptotic cell death could be observed; cell death was thought to be caused by a combination of toxicity due to the effects of the released drug and the effects on the cell membrane of the polymer-bound drug93. These observations can be compared with the intracellular transport and cytotoxicity results that were observed for anti-CD19-targeted liposomal doxorubicin, in which nuclear accumulation of doxorubicin and cell death 760 from apoptosis were observed following rapid internalization of the immunoliposomes and a slow release of the drug from endosomes and lysosomes94,95. The differences might relate to the rate at which each carrier was internalized. A new therapy that combines ADEPT with polymer–drug conjugates — termed PDEPT (polymerdirected enzyme–prodrug therapy — has recently been described96. In prototype experiments, the HPMA copolymer-Gly-Phe-Leu-Gly–doxorubicin (prodrug) was initially administered and allowed to localize by passive targeting to melanoma (B16F10) tumours in mice. This was followed by administration of an HPMA copolymer linked to the enzyme cathespin B, which led to a rapid increase in the rate of doxorubicin release within the tumour. PDEPT resulted in modest increases in lifespan compared with the prodrug alone or with free doxorubicin. The pre-injection of targeted enzyme (or targeted prodrug) might increase the therapeutic effect of the combination. Implications and future directions LTTs have established a toe-hold in the clinic, but clearly there is room for considerable further growth. For much of the past, the LTT technologies have evolved in isolation from the new antibody engineering technologies, and it is only within the last year or two that we have seen the kinds of cross-disciplinary activity that are required to integrate the two approaches. The use of humanized antibodies or fragments, the selection of new, high-affinity targets by phage display and the evolution of improved techniques for linking the ligand with the carrier or therapeutic will all improve the current generation of LTTs. Application of LTT principles to new classes of therapeutics, such as antisense oligonucleotides and gene therapy, promises to lead to advances in applications for these difficult-to-deliver therapeutics. An important issue that is yet to be resolved is the approach that should be used to clinically test LTTs. Evidence in xenograft models indicates that LTTs will be most useful in the treatment of early-stage disease, micrometastases and minimal residual disease. There is clearly a clinical need for new products that are active in an adjuvant setting. However, under current clinical guidelines, it is difficult to test LTT products for these types of application. In adjuvant chemotherapy, clinical trials tend to be longer, more expensive and require more patients than for products tested in advanced refractory disease. Furthermore, it seems that some LTTs might be more effective in combination chemotherapy than as a monotherapy, but a new product must normally prove itself as a monotherapy before it can be tested in combination chemotherapy. It is perhaps not surprising that most successful LTTs are approved for the treatment of haematological diseases, for which large numbers of surface eptiopes have been identified, the target epitopes are expressed on a high percentage of the target cells, and the target cells have a moderate to high degree of accessibility from the vasculature. | OCTOBER 2002 | VOLUME 2 www.nature.com/reviews/cancer © 2002 Nature Publishing Group REVIEWS For most targeted formulations, some mechanism for drug release must exist, with the release preferably triggered once the formulation has reached its target cells or tissues. As therapeutics are generally not biologically active when they are trapped within carriers, attached to polymers or ligands, or present in a prodrug form, the potential exists for substantial improvements in the therapeutic outcome of targeted microreservoir systems, immunoconjugates and ADEPT by optimizing the release rates for the therapeutics.Various release strategies are being explored, including drug release that is mediated by hyperthermia, lysosomal enzymes or pH-sensitive mechanisms that release the drug in response to internalization of the LTT into the acidic, enzyme-rich environment of endosomes and lysosomes87,88,90,97–99. Although preliminary data indicate the potential for these approaches, more research is required in order to understand the basic principles that are involved in designing optimal release rates for different systems, different diseases and/or disease stages, and how these desired release rates can be achieved. 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In vitro and in vivo studies of a VEGF121/rGelonin chimeric fusion toxin targeting the neovasculature of solid tumors. Proc. Natl Acad. Sci. USA 99, 7866–7871 (2002). NATURE REVIEWS | C ANCER Acknowledgements T. Allen’s research into targeted therapeutics is supported by the Canadian Institutes for Health Research and by ALZA Corporation, Mountain View, California, USA. Online links DATABASES The following terms in this article are linked online to: Cancer.gov: http://www.cancer.gov/cancer_information/ acute myeloid leukaemia | breast cancer | chronic myelogenous leukaemia | colorectal cancer | lung cancer | lymphoma | melanoma | non-Hodgkin’s lymphoma | ovarian cancer LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ ABL | BCR | β-lactamase | cathepsin B | CD19 | CD20 | CD22 | CD25 | CD33 | CPG2 | ERBB2 | GM-CSF | IL-2 | MUC1 | tenascin | transferrin Medscape DrugInfo: http://www.medscape.com/druginfo/ cyclophosphamide | daunorubicin | denileukin diftitox | doxorubicin | 5-fluorouracil | gemtuzumab ozogamicin | ibritumomab tiuxetan | imatinib | methotrexate | paclitaxel | rituximab | topotecan | trastuzumab | vincristine FURTHER INFORMATION Cancer.gov Clinical Trials Database: http://www.cancer.gov/search/clinical_trials Protein Reviews on the Web (PROW): http://www.ncbi.nlm.nih.gov/prow Access to this interactive links box is free online. VOLUME 2 | OCTOBER 2002 | 7 6 3 © 2002 Nature Publishing Group ONLINE Biography Theresa Allen is a professor of pharmacology and an adjunct professor of oncology at the University of Alberta in Edmonton, Canada. She has been active in the drug-delivery field for 25 years, and has made important contributions to the development of long-circulating liposomes and targeted liposomal carriers for anticancer drugs and gene medicines. She is a former chair of the Gordon Conference on Drug Carriers in Biology and Medicine and a recent recipient of the Alec Bangham Award for lifetime achievement in the field of liposome research. LocusLink ABL Databases Cancer.gov acute myeloid leukaemia http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=11429 4 http://www.cancer.gov/cancerinfo/pdq/treatment/adultAML/h ealthprofessional/ cathepsin B breast cancer CD19 http://www.cancer.gov/cancerinfo/pdq/treatment/breast/health professional/ http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=930 http://www.ncbi.nlm.nih.gov/LocusLink/list.cgi?Q=abl1%20 or%20abl2&ORG=Hs BCR http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=613 β-lactamase http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=1508 CD20 chronic myelogenous leukaemia http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=931 http://www.cancer.gov/cancerinfo/pdq/treatment/CML/healthprofessional/ CD22 http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=933 colorectal cancer http://www.cancer.gov/cancer_information/cancer_type/colon _and_rectal/ CD25 lung cancer CD33 http://www.cancer.gov/cancer_information/cancer_type/lung/ http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=945 lymphoma CPG2 http://www.cancer.gov/cancer_information/cancer_type/lymphoma/ http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=85448 http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=3559 [link OK?] melanoma http://www.cancer.gov/cancerinfo/pdq/treatment/melanoma/h ealthprofessional/ ERBB2 non-Hodgkin’s lymphoma GM-CSF http://www.cancer.gov/cancerinfo/pdq/treatment/adult-nonhodgkins/healthprofessional/ http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=1437 http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=2064 IL2 ovarian cancer http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=3558 http://www.cancer.gov/cancerinfo/pdq/treatment/ovarianepithelial/healthprofessional/ MUC1 http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=4582 tenascin © 2002 Nature Publishing Group ONLINE http://www.ncbi.nlm.nih.gov/LocusLink/list.cgi?Q=tnc%20or %20tnr%20not%20tnnc1&ORG=Hs transferrin methotrexate http://www.medscape.com/druginfo/Pharm?id=13905&name=METHOTREXATE+ORAL&DrugType=1&Me nuID=PHM&ClassID=N&GeneralStatement=N http://www.ncbi.nlm.nih.gov/LocusLink/LocRpt.cgi?l=7018 paclitaxel http://www.medscape.com/druginfo/Pharm?id=A10166&name=PACLITAXEL+INTRAVEN.&DrugType=1& MenuID=PHM&ClassID=N&GeneralStatement=N Medscape DrugInfo cyclophosphamide http://www.medscape.com/druginfo/Pharm?id=13893&name=CYCLOPHOSPHAMIDE+ORAL&DrugType= 1&MenuID=PHM&ClassID=N&GeneralStatement=N daunorubicin http://www.medscape.com/druginfo/Pharm?id=A3919&name=DAUNORUBICIN+HCL+INTRAVEN.&Drug Type=1&MenuID=PHM&ClassID=N&GeneralStatement=N denileukin diftitox http://www.medscape.com/druginfo/Druginf?id=A-19075&name=DENILEUKIN+DIFTITOX+INTRAVEN.&DrugType=1&MenuID=USEDOS&Clas sID=N&GeneralStatement=N doxorubicin http://www.medscape.com/druginfo/Pharm?id=A3916&name=DOXORUBICIN+HCL+INTRAVEN.&DrugTy pe=1&MenuID=PHM&ClassID=N&GeneralStatement=N 5-fluorouracil http://www.medscape.com/druginfo/Pharm?id=A3907&name=FLUOROURACIL+INTRAVEN.&DrugType=1 &MenuID=PHM&ClassID=N&GeneralStatement=N gemtuzumab ozogamicin http://www.medscape.com/druginfo/Pharm?id=A21218&name=GEMTUZUMAB+OZOGAMICIN+INTRAVEN.&DrugType=1&MenuID=PHM&ClassID=N&GeneralS tatement=N rituximab http://www.medscape.com/druginfo/Druginf?id=A16848&name=RITUXIMAB+INTRAVEN.&DrugType=1& MenuID=USEDOS&ClassID=N&GeneralStatement=N topotecan http://www.medscape.com/druginfo/Druginf?id=A11381&name=TOPOTECAN+HCL+INTRAVEN.&DrugTyp e=1&MenuID=USEDOS&ClassID=N&GeneralStatement=N trastuzumab http://www.medscape.com/druginfo/Druginf?id=A18801&name=TRASTUZUMAB+INTRAVEN.&DrugType= 1&MenuID=USEDOS&ClassID=N&GeneralStatement=N vincristine http://www.medscape.com/druginfo/Druginf?id=A3913&name=VINCRISTINE+SULFATE+INTRAVEN.&Dru gType=1&MenuID=USEDOS&ClassID=N&GeneralStateme nt=N Further information Cancer.gov Clinical Trials Database http://www.cancer.gov/search/clinical_trials Protein Reviews on the Web (PROW) http://www.ncbi.nlm.nih.gov/prow ibritumomab tiuxetan http://www.medscape.com/druginfo/Pharm?id=A23335&name=IBRITUMOMAB-INDIUM-111%2FALBUMIN+HUMAN+INTRAVEN.&DrugType=0&MenuID=PHM &ClassID=N&GeneralStatement=N imatinib http://www.medscape.com/druginfo/Pharm?id=122096&name=IMATINIB+MESYLATE+ORAL&DrugType =1&MenuID=PHM&ClassID=N&GeneralStatement=N © 2002 Nature Publishing Group