Download LIGAND-TARGETED THERAPEUTICS IN ANTICANCER THERAPY

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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.
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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
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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.
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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.
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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
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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.
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‘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
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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
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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.
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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.
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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
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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
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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.
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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.
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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