Download Targeted therapies - Division of Biological and Medical Physics

Professor Yana Reshetnyak, Professor Oleg Andreev and Professor Donald Engelman explain
why their latest research into targeted drug treatments is proving such a success for cancer research
improvements are especially important
for cancer treatment, since the majority of
anticancer drugs are poisons and therefore
toxic for normal cells. While formal toxicity
studies have yet to be done, there is no
indication of any toxicity of pHLIP for cells
or mice. It is possible to target particular cell
surface molecules; however, in many cases it
is not known if these molecules are present
in the tumours of a particular patient, or if
these molecules are present on the surface of a
majority of cells in a tumour. An approach based
on targeting the tumour microenvironment
might be more general.
Firstly, can you provide an overview of the
aims and focus of your research? What are
the central questions driving your project?
What does pHLIP stand for?
Our aims are to elucidate the fundamental
principles of membrane-associated protein
folding and to develop the pHLIP technology
for the imaging and treatment of acidic
diseased tissues. pHLIP (pronounced ‘flip’) is
a family of peptides that we named from ‘pH
(Low) Insertion Peptides’. If the extra cellular
environment is acidic, insertion of these
peptides across a cell membrane is triggered.
The affinity of the peptides for membranes at
low pH is several times higher than at neutral
pH, which results in the targeting of acidic
tissue by pHLIP in vivo.
What is the current state of targeted
drug treatments? In what ways does your
research aim to address the current problems
with targeted cancer drugs?
Specific delivery of therapeutic or imaging
agents to a tumour can be accomplished by
targeting specific molecules overexpressed
on tumour cell surfaces, either using
antibodies or ligands such as vitamins. This
approach has been actively explored for many
years; however, it has some fundamental
limitations. Not all cancer cells have well-
defined biomarkers, and most tumours are
heterogeneous in the expression of a given
marker. Treatment can lead to the selection
of those cells lacking the targeted biomarker,
followed by proliferation of resistant cells.
An important environmental marker present
in almost all solid tumours is acidity. Tumour
acidosis was discovered more than 80 years
ago by Nobel laureate Otto Warburg, but
has been less actively explored as a target
than specific markers. Since pHLIP insertion
is driven by acidity, we had the idea to use it
for targeting cancer cells in solid tumours.
Indeed, pHLIP has demonstrated an excellent
ability to target cells in primary or secondary
(metastasised) tumours. It appears that pHLIP
may be a useful agent for targeting various
types of tumours, working as a theranostic
vehicle that can carry imaging agents,
therapeutic molecules, or both.
Could you explain what obstacles there are
to targeting specific cancer or other disease
cells? How serious a problem are side effects
through targeted drug therapies? How might
pHLIP mitigate these?
Targeted delivery of therapeutic agents to
diseased tissue would produce an increase in
the effective local concentration, reducing
their accumulation in healthy tissue. Such
PROFESSORS RESHETNYAK / ANDREEV / ENGELMAN
Targeted therapies
Could you explain some of the experimental
techniques you have been using? Why have
you chosen specific in vivo models?
We believe the strength of our research
programme is that we are working on all levels –
in vitro, in cultured cells and in vivo, from single
molecules to multiple mice. It allows us to gain
fundamental understanding of the pHLIPsmembrane interaction, tune properties of pHLIP
peptides (currently we have a family of pHLIP
peptides) and test hypotheses on cultured
cells and in vivo. This system provides us with
excellent feedback.
What is meant by a ‘silver bullet’ treatment
and do you consider this something that
could ever become a reality?
Targeting of the tumour microenvironment
could be considered as a more generalised
approach in contrast to receptor-targeting.
However, we do not think that a ‘silver bullet’
is likely to be developed for all cancer types,
and it is beneficial to have a range of imaging
and therapeutic alternatives. But, for aggressive
metastatic solid tumours, we believe that
it should be possible to develop effective
diagnostics and treatments based on their acidic
environment. We believe that a very attractive
approach is to do imaging using various targeted
methods, finding which targeting scheme works
best, and then to use the same targeting scheme
for the delivery of therapeutic agents.
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PROFESSORS RESHETNYAK / ANDREEV / ENGELMAN
On the pHLIP side
With the development of the pHLIP nanotechnology platform, scientists
are on the verge of a breakthrough in the field of cancer imaging and therapy
DESPITE SIGNIFICANT PROGRESS toward
drugs that specifically target protein biomarkers
for certain kinds of cancer cells, there is still no
‘silver bullet’ against cancer. Targeted drug delivery
would allow drugs to preferentially affect diseased
cells, enhancing therapeutic efficacy while
reducing side effects. It is particularly important
for cancer therapy, since most anticancer drugs are
toxic, killing not only cancer cells but also causing
serious damage to healthy cells. Unfortunately,
cells in tumours are heterogeneous and can be
selected for resistance to protein-targeted drugs
and to the immune system.
One of the universal differences between
cancerous and normal tissues is that the former
exhibit a significantly acidic extra cellular
environment. Acidosis is a hallmark of tumour
development both at very early and advanced
stages, as a consequence of both anaerobic
metabolism (the Pasteur effect) and the ‘aerobic
glycolysis’ known as the Warburg effect. As a
result of this phenomenon, the development
of pH-targeted drugs and delivery systems
could lead to a breakthrough in cancer therapy.
To explore this possibility, Professor Yana
Reshetnyak and Professor Oleg Andreev of the
University of Rhode Island have joined forces with
Professor Donald Engelman of Yale to set up a
project directed toward the development of a pH
(Low) Insertion Peptide (pHLIP) nanotechnology
platform for cancer imaging and treatment.
side effects, limiting their efficacy. The researchers’
strategy is to attach pHLIP to these drugs and
make new constructs that will be minimally
active in normal tissue, but delivered into cells in
a diseased acidic environment, thus reducing side
effects. Liposomes have proven highly effective
transporters of various therapeutic materials, and
preliminary data indicates that pHLIP can guide
liposomes to tumours and promote their uptake
by cells, releasing payloads into various cellular
compartments. A pHLIP-liposome delivery system
would enable a larger range of delivered molecules,
including enzymes, and even assist in the selective
delivery of gene regulating agents such as DNA,
siRNA and Peptide Nucleic Acids (PNAs).
TARGETED DELIVERY OF POLAR MOLECULES
The
most
revolutionary
and
important
application of the pHLIP technology is
the targeted delivery of polar, biologically
functional molecules that ordinarily cannot
cross membranes. We have shown that polar
molecules (including toxins) can be delivered
by pHLIP into cultured cells in acidic extra
cellular environments and released into the
cytoplasm by disulfide bond cleavage, where
they induce inhibition of cancer cell growth. This
strategy would significantly expand the range
of therapeutics and improve the specificity in
comparison with existing drug therapies. The
most important application will be the treatment
of metastatic cancers as pHLIP targets not only
primary tumours but also metastases as small
as 1 mm. With few treatment options available
to patients with metastatic cancer, it is hoped
there might be an accelerated path for approval.
The team are currently testing delivery of several
The original pHLIP was discovered in folding
studies of the third transmembrane helix from
bacterorodopsin. We have now designed a
family of 25-40 amino acids moderately polar
membrane peptides that bind to the surfaces of
cell membrane phospholipid bilayers at neutral
pH. If the extracellular environment is acidic,
protonation of Asp/Glu residues occurs, which
enhances peptide hydrophobicity and triggers
peptide insertion across the cell membrane. The
affinity of the peptides for membranes at low pH
is several times higher than at neutral pH, which
results in the targeting of acidic tissue by pHLIP in
vivo. The project seeks to develop the technology
for use in two ways: pH-selective targeting of
therapeutic or imaging agents to solid tumours,
where they are tethered to the surfaces of cancer
cells; and pH-selective targeting of tumour cells
with cytoplasmic delivery of cargo molecules
attached to the pHLIP via a cleavable bond.
THERAPEUTIC DELIVERY
pHLIP is a platform technology that allows
selective delivery of diagnostic and/or therapeutic
agents including immune activation molecules
to acidic tissues. The project has been testing
whether this technology could be used for the
delivery of drugs that are currently approved for
clinical use. The majority of approved anticancer
drugs are not very selective and induce significant
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INTERNATIONAL INNOVATION
FIGURE 1. Whole-body NIR (Alexa750-pHLIP), GFP fluorescence and light images of mouse bearing tumour are shown.
Highly metastatic M4A4 cells derived from the human melanoma cancer cell line, MDA-MB-435, were used to establish the
tumour. Fluorescently labelled pHLIP (Alexa750 was covalently attached to the Cys residue at the N-terminus of peptide) was
given as a single iv injection (0.8 mg/kg). The images were obtained on the FX Kodak in vivo image station combined with a
gas anaesthesia system at 4, 24, 48 and 72 hours post-injection. Tumour indicated by arrows. Tumour targeting was observed
already after four hours after injection of fluorescent pHLIP, and peptide stays in tumour during several days. Images obtained
at various time points presented with maximum contrast between tumour and other tissue. Taken from Chemistry Today.
polar compounds by pHLIP, which might be
candidates for new anticancer drugs.
ENHANCING IMAGING TECHNIQUES
In addition to its potential for targeted
administration of drugs, pHLIP also has a valuable
part to play in improving current surgical techniques
for the removal of cancers via the assistance of
image guided surgery. pHLIP can be used to deliver
agents for fluorescence, PET, SPECT and, potentially,
MR imaging. Over time these agents accumulate in
tumours, thus enhancing the contrast index and
reducing the presence of the agent in healthy tissue.
The higher the contrast index, the more accurate
the imaging becomes.
The recurrence of certain cancers remains quite
high due to either incomplete surgical removal
of the primary tumour or the presence of small
metastases that are invisible to the surgeon. Near
infrared (NIR) fluorescence imaging might improve
surgical outcomes by providing sensitive, specific,
and real-time visualisation of normal and diseased
tissues using agents such as pHLIP that discriminate
between normal and diseased tissue and define
tumour margins. The team has demonstrated that
the injection of pHLIP molecules conjugated with
an NIR dye results in the fluorescent visualisation
of tumours in mice. Tumour boundaries are well
marked, and small fragments (<1 mm) can be
visualised, which are usually imperceptible to the
eye. Fluorescently labelled pHLIP can help in the
effective removal of all diseased tissues and increase
the success of surgery.
FURTHER EXPLORATION
The project has not been without challenges along
the way. For example, all normal tissues and organs
have pHs in the range of 7.2-7.4, except the kidney;
therefore, pHLIPs, whilst targeting diseased tissue,
also target the kidneys. This may make no difference
for applications in imaging but it could be a potential
problem for the delivery of toxic agents. One
possible solution to this is local activation therapy.
The concept is to accumulate an activatable agent
in the tumour using pHLIP. The construct would
be injected, and later the therapy would be locally
activated to destroy tumour tissue. To explore this
possibility, clusters of gold atoms were delivered
to tumours. Irradiation of gold particles with soft
x-rays (which cause little damage to tissues and
require only inexpensive radiation sources) results
in the generation of reactive species including Auger
electrons that kill only nearby cells. Other kinds of
local activation by infrared light or magnetic fields
could also be applied using other targeted entities.
The main purpose of such a technique is to limit
damage to the actual cancer cells, and to treat
only the local region with the activating stimulus.
So although pHLIP targets both implanted and
spontaneous tumours very well, the presence of
other acidic tissues, such as the kidney, might favour
local activation therapy.
In addition, acidity is not only characteristic of
tumours but also other pathological states such as
ischemia, stroke, infarction, inflammation, arthritis,
and injury, suggesting the pHLIP-delivery platform
may have broad applicability. Since acidity is a
common feature of various pathological states,
it might be possible to use pHLIP as a ‘universal
marker for diseased tissues’. Using pHLIP conjugated
with an appropriate imaging agent the entire
body can be scanned and diseased spots could be
detected. The pHLIP test can be used as a routine
general health check, which would allow diseases
to be identified at a much earlier stage, even before
the patient feels any pain. The team expects that
the first pHLIP-imaging product, currently being
produced by a major company, will be developed
for clinical use within the next few years. Other
imaging pHLIP products might be developed in the
near future, although further testing is still required
for therapeutic pHLIP based products.
FIGURE 2. Fluorescent pHLIP targets metastatic nodules in lungs and is distributed in the extracellular space and cellular
membranes of the tumour cells. A
primary tumour was established by
subcutaneous injection of M4A4
cancer cells expressing GFP, and the
tumour was grown until it gave lung
metastases. Then, the primary tumour
was removed and Alexa750-pHLIP
was given as a single iv injection.
One day after injection, the animal
was euthanised, the chest was
opened, and whole-body imaging was
carried out. a) Whole-body GFP, NIR
(Alexa750-pHLIP) fluorescence and
visible light (photo) images are shown.
b) Co-localisation of GFP and NIR
fluorescence is shown in the excised
lungs. c) A metastatic lesion analysed
under the fluorescence microscope at
10x magnification demonstrates colocalisation of GFP and NIR emission. d)
A detailed analysis of NIR (Alexa750pHLIP) fluorescence distribution was
carried at 100x magnification. The
NIR fluorescence is distributed in the
extracellular space (*) with staining of
the cellular membrane (indicated by
arrows). Taken from Journal of Molecular
Imaging and Biology.
INTELLIGENCE
PHLIP NANOTECHNOLOGY PLATFORM
FOR CANCER IMAGING AND THERAPY
OBJECTIVES
pHLIP is a platform technology that allows
selective delivery of diagnostic and/or
therapeutic agents to acidic tissues. Acidity is
characteristic of tumours and other pathological
states such as ischemia, stroke, infarction,
inflammation, arthritis, and injury, so the pHLIPdelivery platform may have broad applicability.
KEY COLLABORATORS
Professor Donald M Engelman, Higgins
Professor of Molecular Biophysics, Yale
University • Professor Oleg A Andreev,
Associate Professor, University of Rhode Island
FUNDING
NIH – NCI
CONTACT
Yana K Reshetnyak, PhD, Associate Professor
Division of Biological and Medical Physics
Department of Physics, University of Rhode Island
East Hall 223, 2 Lippitt Road
Kingston, RI 02881-0817, USA
T +1 401 874 2060
E [email protected]
www.biophys.phys.uri.edu
DONALD M ENGELMAN is the Higgins
Professor of Molecular Biophysics at Yale. He
has a BS in Physics from Reed College, a PhD in
Biophysics from Yale, and Postdoctoral training
at UCSF and the University of London, King’s
College. He is an author of more than 200
scientific publications, which have been cited
more than 15,000 times.
YANA K RESHETNYAK is a tenured Associate
Professor at the University of Rhode Island. She
has an MS in Molecular Physics from the SaintPetersburg State University, and she obtained
her PhD in Biophysics in Pushchino, Russian
Academy of Science, followed by postdoctoral
work in the Cancer Center in Texas, and in the
Engelman laboratory at Yale, where she started
to develop the pHLIP technology.
OLEG A ANDREEV is a tenured Associate
Professor at the University of Rhode Island. He
obtained his MS in Physics and PhD in Biophysics
from the Moscow Institute of Physics and
Technology (MIPT). In 1990 he came to the U.S.
to continue muscle research at the Baylor Medical
Center in Dallas, the University of Texas at Austin,
and later at the University of North Texas as a
Research Assistant Professor and Director of the
Microscopy and Spectroscopy Laboratory.
AND YALE
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