Tumor-Penetrating Peptides

Cancer Research Center, Sanford-Burnham Medical Research Institute , La Jolla, CA , USA
Frontiers in Oncology 08/2013; 3:216. DOI: 10.3389/fonc.2013.00216
Source: PubMed
Tumor-homing peptides can be used to deliver drugs into tumors. Phage library screening in live mice has recently identified homing peptides that specifically recognize the endothelium of tumor vessels, extravasate, and penetrate deep into the extravascular tumor tissue. The prototypic peptide of this class, iRGD (CRGDKGPDC), contains the integrin-binding RGD motif. RGD mediates tumor-homing through binding to αv integrins, which are selectively expressed on various cells in tumors, including tumor endothelial cells. The tumor-penetrating properties of iRGD are mediated by a second sequence motif, R/KXXR/K. This C-end Rule (or CendR) motif is active only when the second basic residue is exposed at the C-terminus of the peptide. Proteolytic processing of iRGD in tumors activates the cryptic CendR motif, which then binds to neuropilin-1 activating an endocytic bulk transport pathway through tumor tissue. Phage screening has also yielded tumor-penetrating peptides that function like iRGD in activating the CendR pathway, but bind to a different primary receptor. Moreover, novel tumor-homing peptides can be constructed from tumor-homing motifs, CendR elements and protease cleavage sites. Pathologies other than tumors can be targeted with tissue-penetrating peptides, and the primary receptor can also be a vascular "zip code" of a normal tissue. The CendR technology provides a solution to a major problem in tumor therapy, poor penetration of drugs into tumors. The tumor-penetrating peptides are capable of taking a payload deep into tumor tissue in mice, and they also penetrate into human tumors ex vivo. Targeting with these peptides specifically increases the accumulation in tumors of a variety of drugs and contrast agents, such as doxorubicin, antibodies, and nanoparticle-based compounds. Remarkably the drug to be targeted does not have to be coupled to the peptide; the bulk transport system activated by the peptide sweeps along any compound that is present in the blood.


Available from: Tambet Teesalu, Oct 14, 2014
published: 27 August 2013
doi: 10.3389/fonc.2013.00216
Tumor-penetrating peptides
Tambet Teesalu
, Kazuki N. Sugahara
and Erkki Ruoslahti
Cancer Research Center, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA
Laboratory of Cancer Biology, Centre of Excellence forTranslational Medicine, Institute of Biomedicine and Translational Medicine, University of Tartu,Tartu, Estonia
Department of Surgery, College of Physicians and Surgeons, Columbia University, NewYork, NY, USA
Department of Cell, Molecular and Developmental Biology, University of California Santa Barbara, Santa Barbara, CA, USA
Edited by:
Angelo Corti, San Raffaele Scientific
Institute, Italy
Reviewed by:
Angelo Corti, San Raffaele Scientific
Institute, Italy
Fabrizio Marcucci, Istituto Superiore
di Sanità, Italy
Erkki Ruoslahti, Cancer Research
Center, Sanford-Burnham Medical
Research Institute, 10901 NorthTorrey
Pines Road, La Jolla, CA 92037, USA
Tumor-homing peptides can be used to deliver drugs into tumors. Phage library screen-
ing in live mice has recently identified homing peptides that specifically recognize the
endothelium of tumor vessels, extravasate, and penetrate deep into the extravascular
tumor tissue. The prototypic peptide of this class, iRGD (CRGDKGPDC), contains the
integrin-binding RGD motif. RGD mediates tumor-homing through binding to αv integrins,
which are selectively expressed on various cells in tumors, including tumor endothelial
cells.The tumor-penetrating properties of iRGD are mediated by a second sequence motif,
R/KXXR/K. This C-end Rule (or CendR) motif is active only when the second basic residue
is exposed at the C-terminus of the peptide. Proteolytic processing of iRGD in tumors
activates the cryptic CendR motif, which then binds to neuropilin-1 activating an endocytic
bulk transport pathway through tumor tissue. Phage screening has also yielded tumor-
penetrating peptides that function like iRGD in activating the CendR pathway, but bind to
a different primary receptor. Moreover, novel tumor-homing peptides can be constructed
from tumor-homing motifs, CendR elements and protease cleavage sites. Pathologies other
than tumors can be targeted with tissue-penetrating peptides, and the primary receptor
can also be a vascular “zip code of a normal tissue. The CendR technology provides a
solution to a major problem in tumor therapy, poor penetration of drugs into tumors. The
tumor-penetrating peptides are capable of taking a payload deep into tumor tissue in mice,
and they also penetrate into human tumors ex vivo. Targeting with these peptides specifi-
cally increases the accumulation in tumors of a variety of drugs and contrast agents, such
as doxorubicin, antibodies, and nanoparticle-based compounds. Remarkably the drug to be
targeted does not have to be coupled to the peptide; the bulk transport system activated
by the peptide sweeps along any compound that is present in the blood.
Keywords: synaphic targeting, homing peptide, tumor-penetrating peptide, neuropilin-1, αv integrins, C-end Rule
A major problem in systemic therapy is that only a small propor-
tion of administered drug reaches its intended target site(s). Selec-
tive delivery of the drug to the target tissue can alleviate this prob-
lem. Affinity-based physical targeting (synaphic, pathotrophic, or
active targeting) makes use of molecular markers that are specif-
ically expressed at the target, and not elsewhere in the body, to
accomplish selective targeting of systemically administered drugs
(1). The desired outcome of the synaphic targeting is similar
to topical application: increased local accumulation and lower
systemic concentration of the therapeutic payload.
Synaphic targeting efforts have led to improved cancer drug
delivery, but this approach only partially solves the selective
delivery problem. Delivering a payload to a molecule specif-
ically expressed on the surface of vascular cells in the target
tissue can be effective because the vasculature is readily avail-
able for blood-borne probes. Thus, anti-angiogenic and vascular
disrupting compounds can benefit from this approach. In fact,
many of these compounds inherently target the vascular endothe-
lium. An obvious example is antibodies that block the vascular
endothelial growth factor receptors [VEGF-Rs, (2)]. These recep-
tors are generally expressed at elevated levels in tumor vasculature.
Hence the antibody (or other VEGFR ligand) has more binding
sites in tumor vessels than elsewhere and could selectively carry
a payload there. Less well known is that many of the natural
and designed anti-angiogenic proteins highjack integrin-binding
plasma proteins (fibronectin, vitronectin, fibrinogen) to selec-
tively target the angiogenic tumor vessels. The anti-angiogenic
proteins for which this has been shown include angiostatin, endo-
statin, anginex, and anastellin (3). However, besides tumor ves-
sels, it is desirable to also target the tumor cells (and stromal
cells) within the tumor. While delivering a drug to tumor ves-
sels can improve the efficacy of the drug, the drug still has to
extravasate and penetrate into the extravascular tumor tissue to
reach the tumor cells. The technology we review in this article
provides a solution to the tumor penetration problem. It can also
help to deal with another, less appreciated problem of synaphic
delivery: that the number of available receptors in a tumor is
likely to be too low for the delivery of sufficient quantities of a
payload drug. August 2013 | Volume 3 | Article 216 | 1
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Teesalu et al. Tumor-penetrating peptides
The endothelia of vessels in different organs, even when mor-
phologically indistinguishable, are molecularly (and as a result,
likely functionally) different [“vascular zip codes, (4)]. Moreover,
specific response patterns are activated in vascular cells during
processes such as tumor growth, inflammation, tissue repair, and
atherosclerosis. Many of the zip codes elicited by these processes
are secondary to angiogenesis, the sprouting of new blood vessels
from existing vessels. A common denominator is endothelial cell
(and pericyte) activation, but each condition can also put an indi-
vidual signature of the vasculature. One set of activation-related
cell surface molecules, comprised of P-selectin, E-selectin, ICAM-
1, and VCAM-l, is turned on by inflammation in venular endothe-
lial cells and mediates leukocyte rolling and adhesion/emigration
in response to inflammation (5, 6). Another signature set of cell
surface molecules, comprising certain integrins, growth factor
receptors, extracellular proteases, and extracellular matrix pro-
teins, is expressed during angiogenesis, which is the main factor
making tumor vasculature distinguishable from normal vascula-
ture in the adult organism. Lymphangiogenesis and macrophage
infiltration also contribute to tumor-related marker molecules (7).
In vivo phage display has been instrumental in establishing
the extent of the molecular specialization in the vasculature and
has contributed a number of new markers of tumor vasculature
(4, 8). Bacteriophage can be genetically modified to incorporate
random peptide sequences as fusions with the coat proteins at a
diversity of about one billion variants per library, which is close to
the total number of possible permutations of a random 7-amino
acid sequence (1.28E9). For in vivo selection, a library of phage
displaying random peptides is injected systemically into the ani-
mals, followed by removal of target organs, amplification of the
bound phage, and subjecting the amplified pool to another round
of selection. In vivo peptide phage screening combines subtrac-
tive elements (removal of phage displaying pan-specific peptides)
with positive selection at the target tissue (9). This technology
has yielded peptides with unique tumor-penetrating properties as
discussed below.
About 10 years ago, our laboratory identified a peptide, LyP-1
(CGNKRTRGC), with the ability to take the phage expressing
it to the lymphatic vessels and hypoxic areas in tumors (10,
11). Surprisingly, the LyP-1 phage reached its targets in tumors
within minutes of intravenous injection. Given that the phage is
a nanoparticle and consequently diffuses slowly, diffusion did not
seem to account for the rapid spreading within the tumor. It took
the discovery of the CendR system, and the realization that it was
responsible for the spreading within tumors of a more recently
identified tumor-homing peptide, iRGD, to understand how these
peptides penetrate into tumors (12, 13).
Tumor-penetrating peptides like iRGD and LyP-1 contain three
independent modules: a vascular homing motif, an R/KXXR/K
tissue penetration motif, and a protease recognition site. These
modules cooperate to ensure a multistep, highly specific process
of tumor-homing and penetration. The sequence of the prototypic
tumor-penetrating peptide, iRGD, is CRGDR/KGPDC. We mostly
use the K-variant, CRGDKGPDC, because it appears to provide
stronger tumor-homing than the R-variant. Following systemic
administration, the iRGD peptide is first recruited through its
RGD motif to αv integrins, which are overexpressed on tumor
endothelial cells. After the initial binding, proteolytic process-
ing exposes the internal R/KXXR/K motif at the C-terminus of
the truncated peptide. We have termed the R/KXXR/K motif
the C-end Rule or CendR motif (pronounced sender) because
of the requirement of C-terminal exposure for activity. The C-
terminal CendR motif interacts with neuropilin-1 (NRP-1), and
the NRP-1 interaction triggers the activation of a transport path-
way (CendR pathway) through the vascular wall and through
extravascular tumor tissue (12, 13). These peptides can take along
both conjugated and co-administered payloads into the tumor
We came across the CendR phenomenon while screening phage
libraries for peptides that would bind to and internalize into cells
isolated from tumors grown in mice. We were initially disap-
pointed to find that, independent of the starting library con-
figuration (we used cysteine-flanked cyclic and linear random
heptapeptide libraries), the selected peptides all looked similar;
they all had a C-terminal arginine or lysine residues with another
basic amino acid at the 3 position. However, we soon realized
that the consensus motif, R/KXXR/K, had to be some kind of a
master cell internalization signal and set out to study it. It is worth
noting that, while our laboratory used the filamentous phage dis-
play system introduced by Smith (14, 15) in our early studies (8,
16), we later switched to the T7 phage. The important distinction is
that in T7, the exogenous peptide is expressed at the C-terminus of
the phage coat protein, whereas it is at the N-terminal end in the
filamentous phage. Thus, the C-terminal truncations producing
the CendR motif could only be selected for in the T7 system.
The binding and internalization of R/KXXR/K-displaying
phage or synthetic nanoparticles required the presence of free
C-terminal arginine or lysine residues as addition of additional
amino acid residues to the motif or amidation of the carboxyl ter-
minus resulted in loss of activity (12). In addition to the prostate
cancer cell lines, the active CendR motif triggered binding, and
internalization in many cultured tumor cell lines and in cells in
suspensions prepared from normal mouse tissues. Studies on the
prototypic active CendR peptide,RPARPAR, showed that the bind-
ing only takes place for the peptide made of l-amino acids and that
the binding can be inhibited by excess of free peptide, suggest-
ing the existence of a saturable receptor with a chiral recognition
specificity. In contrast, cell-penetrating peptides, widely used for
intracellular delivery of payloads in vitro are independent of posi-
tion and chirality, and no specific receptors for them have been
Affinity chromatography with RPARPAR identified NRP-1 as
the main binding molecule for RPARPAR. NRP-1 is a trans-
membrane receptor with major roles in cell migration and
endothelial cell sprouting in blood vessels, while NRP-2 with a
similar, but not identical binding specificity is abundant and plays
an important role in lymphatic vessels (17, 18). NRP-1 is best
known for its role as a co-receptor for certain members of the vas-
cular endothelial growth factor (VEGF) and semaphorin families
(19, 20). The NRP-1-binding VEGFs and semaphorins, and TGFβ,
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Teesalu et al. Tumor-penetrating peptides
all have C-terminal CendR motifs. Tuftsin is an immonomodu-
latory peptide that has been shown to bind to NRP-1 [it has a
C-terminal arginine residue, but lacks the complete CendR motif;
(21)]. It induces vascular permeability (22), but no evidence on
tissue penetration has been presented.
The b1b2 domain of NRP-1 that contains the binding pocket
for the CendR motif has been crystallized together with tuftsin
(23). Molecular modeling studies show that peptides with a C-
terminal CendR motif, such as RPARPAR fit well to the binding
pocket,but do not provide an explanation for the role of the penul-
timate arginine residue, which remains outside the binding pocket
[Figure 1; (24, 25)]. Perhaps this arginine could be engaging an
as-yet unknown molecule in a three-way interaction with NRP-1.
Based on molecular simulations and phage binding to puri-
fied NRP-1 protein it appears that formation of a stable complex
between a CendR peptide and NRP-1 requires interaction of the
-2 and -3 residues with loop III of the b1 domain of the NRP-1,
as in the case of RPAR, RRAR, RDAR, RPDR, RPRR, and RPPR
(25). For a stable interaction to occur, loop III must be engaged
in a pairwise interaction that stabilizes the interaction of the C-
terminal carboxylic group with the CendR binding pocket in the
b1 domain of NRP-1.
Interestingly, the D-conformer of RPARPAR is a poor fit with
the binding pocket, suggesting that the D-Tat, even with a C-
terminal arginine would not bind to NRP-1. The modeling studies
also indicate that under some circumstances a cyclic peptide could
fit into the binding pocket (24). Indeed, peptides built on a ther-
mostable, protease-resistant cyclotide kalata B1 scaffold have been
described that are thought to interact with NRP-1 as intact cyclic
peptides (26). These modeling studies provide a basis for in sil-
ico screening of CendR analogs and evaluation of low molecular
FIGURE 1 | Ribbon representation of the NRP-1-RPAR complex
showing the most notable interactions found between the peptide
and the binding pocket of NRP-1. The ligand and the interacting side
chains of the receptor are depicted as solid lines. NRP-1 backbone is shown
in purple and RPAR backbone in green Hydrogen atoms are omitted for
clarity. Specific interactions are drawn: hydrogen bonds are shown as blue
discontinuous lines while salt bridges are marked by yellow discontinuous
lines. Reprinted with permission from Haspel et al. (24). Copyright 2011
American Chemical Society.
weight compounds resulting from high throughput screening. The
molecules that bind to the CendR binding pocket on b1b2 domain
of NRP-1 will be either acting as agonists or antagonists with
potential applications in cancer drug delivery, and in diseases asso-
ciated with elevated vascular permeability and pathogen spreading
in tissues (see below).
A wide range of other receptors have been reported to use NRP-
1 as a co-receptor, earning NRP-1 designation as a “hub receptor
(27), but it is not clear whether the ligands of these receptors use
the CendR motif binding site for docking to NRP-1. Whereas NRP-
1 can signal independently of other signal-transducing receptors,
the primary role of NRP-1 is believed to be acting as a co-receptor
that ensures the recruitment and presentation of various ligands
to the effector receptors. NRP-1 is overexpressed in many can-
cer cell lines, where it is implicated in migration, proliferation,
and survival. NRP-1 is overexpressed in tumors, both in cancer
cells and in stromal cells, and is implicated in development and
maintenance of the tumor vessels and in tumor growth and pro-
gression (28, 29). NRP-1 is a target of anti-cancer therapy with
antibodies and peptide-bound therapeutic agents (3034). How-
ever, as the NRPs are also widely expressed in normal vessels,
the overexpression in tumors will only afford a degree of tumor
specificity. Another aspect is that in bloodstream, plasma pro-
teases carboxypeptidases [e.g., carboxypeptidase M and N; (35)]
rapidly remove C-terminal arginine residues,thus limiting the effi-
cacy of systemic active CendR peptides in tumor drug delivery. In
contrast, the localized tumor-specific proteolytic activation of the
cryptic CendR motif of our tumor-penetrating peptides results in
tumor-specific activation of a cell and tissue penetration pathway.
The ability of VEGF and semaphorins to increase vascular per-
meability has been recognized for some time. Dvorak and Feng
(36) showed that VEGF induces the formation of a network of
tubular vesicles in endothelial cells they named the “Vesiculo-
vacuolar organelle, and presented morphological evidence that
these interconnected vesicles could form a pathway though cells.
The complicating factor in interpreting these results is the activ-
ity of the main signaling receptors for VEGF (VEGF-Rs) and for
the semaphorins (plexins). CendR peptides allow one to study the
NRP binding in isolation of other receptors and have made it pos-
sible to show that NRP-1 [and NRP-2, (37)] activate a trans-tissue
transport pathway.
The uptake of the payload of CendR peptides into intracellular
vesicles shows that the entry into cells is through an endosomal
route. Moreover, the rapid penetration of the payloads of tumor-
homing CendR peptides into tumors in vivo and ex vivo, and
its energy dependence (13, 37, 38) shows that this is an active
transport pathway, not one dependent on diffusion. The CendR
pathway may be distinct from the known endosomal pathways,
but at this point the evidence to that effect is limited to the use
of various pharmaceutical inhibitors of the known pathways (12).
The extravasation and tumor-penetration activities of iRGD sug-
gest that the payload of the CendR endocytic vesicles is also at
least partially released from cells by fusion of the endosomes with
the plasma membrane. We have not yet observed the exocytosis
phase of this presumed transcellular pathway, but the rapid tissue August 2013 | Volume 3 | Article 216 | 3
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Teesalu et al. Tumor-penetrating peptides
penetration of the CendR payloads support of this possibility.
However, we cannot exclude that an alternative pathway such as
propelling cell surface-bound payload forward by the cell mem-
brane or membrane projections. Genetic and proteomics studies
are underway to elucidate the cellular molecular basis of the CendR
Our discovery of the CendR tissue transport pathway raises the
fascinating question of the physiological function of this path-
way. While the focus so far has been on how this pathway might
be used in drug delivery, it obviously does not exist for this pur-
pose. One possibility is that it facilitates the transfer of nutrients
to cells that are far from blood vessels or otherwise under duress.
The overexpression of NRP-1 in tumors suggests that supplying
nutrient deficient/hypoxic areas in tumors may be yet another way
tumors make use of a physiological pathway to foster their own
growth. The CendR pathway may have been hijacked by viruses
and microbial toxins for cell entry and tissue spreading. Cleavage
of a viral surface proteins and pro-toxins by host proteases (most
commonly furins and related enzymes) at sites that create an active
CendR motif is a recurrent theme seen in many pathogens. Exam-
ples include the Human T-lymphotropic virus-2, Crimean–Congo
hemorrhagic fever virus, tick-born encephalitis virus, and Ebola
viruses, as well as anthrax toxin (3943). CendR sequences are
also present in snake and bee venoms (e.g., mellitin), and may
contribute to the spreading of these toxins in tissues.
Vascular edema is associated with many diseases (hemorrhagic
virus infections, sepsis, and vascular leak syndromes). Several
proinflammatory vasoactive (poly)peptides capable of increas-
ing vascular permeability display a functionally important argi-
nine residue at their C-terminus. Examples include complement
C3a and C5a anaphylatoxins (C-terminal sequences ASHLGLAR
and KDMQLGR, respectively) as well as kinins (bradykinin and
kallidin, which have an identical C-terminal sequence, RPPGF-
SPFR). Intriguingly, we have observed that phage that display
peptides corresponding to the C-terminal amino acids of C5/3a
and bradykinin bind to the recombinant b1b2 domain of NRP
(in preparation) and that the binding is reversed by an excess of
the free peptide. It remains to be seen whether the NRP/CendR
axis plays a role in the activity of C3/5a, bradykinin, and/or other
vasoactive peptides.
Having worked out the two-motif requirement for a tumor-
homing peptide to have CendR activity, we tested the universality
of the concept by designing a new peptide with such activities.
We used as the starting point the NGR tumor-homing motif pre-
viously identified by our laboratory (44, 45), which recognizes a
form of aminopeptidase N in angiogenic tumor vessels (46, 47).
We added a second arginine to the NGR motif to convert it into the
CendR motif, RNGR and embedded that motif in the iRGD frame-
work by replacing RGDK with RNGR. The resulting peptide, iNGR
(CRNGRGPDC) has all the properties of iRGD, except that its
tumor recruitment is not mediated by integrin but another recep-
tor, presumably aminopeptidase N (48). We have also designed
tumor-homing CendR peptides by arranging in tandem a CendR
motif, a proteolytic cleavage site for a tumor-associated protease
that cleaves after a basic residue, and a tumor-homing motif
(Teesalu et al., in preparation). These peptides also home to and
penetrate into tumors. A construct created to deliver a non-specific
cell-penetrating peptide, appears to serendipitously follow this
design (49). Whether these tandem tumor-penetrating peptides
are as effective as the peptides in which the homing motif and
CendR motif overlap remains to be seen. The iRGD and LyP-1
peptides lose their affinity for the primary receptor [αv integrins
for iRGD and a mitochondrial/cell surface protein p32 for LyP-1
(7) after the proteolytic cleavage that activates the CendR motif has
taken place (13, 37)]. The resulting release of the peptide from the
primary receptor may facilitate subsequent binding to NRP-1 and
make the primary receptor available for binding of another intact
peptide. Peptides with tandem motifs would lack this latter feature.
Another possible design for CendR activation would be blocking
the C-terminus with a chemical group other than an amino acid or
peptide. One can envision peptides, the CendR activity of which
is triggered by a phosphatase, demethylase, sulfatase, etc. To the
extent such an enzyme is specific for the target tissue, new useful
probes could be created.
To reach tumor cells and tumor-associated parenchymal cells (e.g.,
tumor-associated fibroblasts, macrophages), drugs must cross the
vascular barrier and penetrate into the extravascular stroma. Can-
cerous tissue is heterogeneous, with striking regional differences
in tumor structure (leaky vasculature and defective lymphatics,
which causes buildup of interstitial fluid pressure in the tumor),
and physiology (e.g., inflammation, fibrosis, hypoxia, acidity).
These features translate into steep drug gradients and variabil-
ity in the uptake and distribution of anti-cancer drugs within
tumor parenchyma (50). For example, evaluation of doxorubicin
distribution in tumors after systemic administration showed that
the concentration of this drug decreases exponentially with dis-
tance from tumor blood vessels, reaching half of its perivascular
concentration at a distance of about 40 µm (51). The distribu-
tion of trastuzumab (Herceptin) in breast tumor xenografts is
also highly heterogeneous with many tumor cells exposed to no
detectable drug (52). To some extent,the tumor drug delivery chal-
lenges are alleviated by the Enhanced Permeability and Retention
(EPR) effect – accumulation of compounds (typically liposomes,
nanoparticles, and macromolecular drugs) in tumor tissue more
than they do in normal tissues. The underlying causes of the EPR
effect are abnormal structure and function of tumor vessels: poorly
aligned endothelial cells with fenestrations, deficient pericyte cov-
erage, and lack of lymphatic drainage. However, EPR is highly
variable as it is influenced by differences between tumor types and
heterogeneity within individual tumor. Tumor interstitial pressure
(IFP) depends on integrity of blood and lymphatic vessels, tumor
cell proliferation, deposition of matrix molecules, and interac-
tion of cells with the matrix molecules. The difference between
tumor microvascular fluid pressure and IFP determines intratu-
moral convection fluxes that have a major influence on the vascular
exit of the compounds over 10 kDa. Intratumoral fluid pressure
gradients can be in some cases favorably influenced by vasodilatory
compounds such as bradykinin, endothelin, and calcium channel
antagonists, to allow better tumor perfusion and increased drug
delivery (53). Other approaches include dissolving extracellular
matrix with enzymes such as collagenase or hyaluronidase (54),
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Teesalu et al. Tumor-penetrating peptides
or killing or inhibiting the activity of tumor-associated fibroblasts
(55). Obviously, the delivery of enzymes and drugs aimed at low-
ering the IFP to the tumor parenchyma faces the similar tumor
penetration challenges seen for the cancer drugs.
The tumor-homing CendR peptides provide a solution to the drug
penetration problem. A probe or drug attached to iRGD or LyP-
1 is delivered to extracellular tumor tissue more effectively than
the drug alone. We have extensively demonstrated the tumor pen-
etration with fluorescein (FAM)-labeled peptides. Intravenously
injected FAM-iRGD, LyP-1, and iNGR are found dispersed in
tumor parenchyma minutes after administered, whereas FAM-
labeled inactive control peptides do not appear in the tumors at
all. FAM-labeled homing peptides that lack a CendR motif bind to
the blood vessels, but do not penetrate into the rest of the tumor
(10, 11, 13, 48). Remarkably, iRGD and LyP-1 have quite different
distributions within tumors, presumably reflecting the expression
of their primary receptors in different tumor compartments (7,
10, 13). The effect of the cryptic CendR motif is vividly illus-
trated by the differences between iRGD and conventional RGD
peptides, such as CRGDC and cycloRGDfK. While iRGD pay-
load, even a poorly diffusing nanoparticle, readily enters tumor
parenchyma, the conventional RGD peptides only take their pay-
load to the tumor vessels (13, 38). LyP-1 and CGKRK, a peptide
we have recently shown to also use p32 as its receptor but lack the
CendR activity (56) show a similar difference (11, 57).
The observations with the fluorescent probe described above
prompted us to study the ability of iRGD and the other CendR
peptides to enhance the delivery of actual anti-cancer drugs to
tumors. We have shown that therapeutics as diverse as a small
molecular weight drug (doxorubicin), trastuzumab (anti-Her2
antibody), and the nanoparticle drugs Abraxane and Doxil can
benefit from iRGD-enhanced delivery (13, 38). In showing this,
we mostly made use of a unique property of iRGD and other simi-
lar peptides; they can enhance tumor penetration of payloads that
are not attached to the peptide, just administered at the same time.
The reason is that iRGD activates a bulk transport pathway that
moves along any compound present in the blood when the system
is active. The scheme in Figure 2 illustrates this principle.
Timing measurements have shown that the CendR pathway
is active for about 1 h, with peak activity about 30 min after the
administration of the peptide (38). The timing agrees with the
half-life of the peptide in the blood, which for a peptide of this
size can be expected to be about 10 min (58). The main reason for
the short half-life is elimination of the peptide through filtration
into the urine. It remains to be determined whether prolonging
the half-life of the peptide would further enhance drug delivery
into tumors. We compared the efficacy of directly conjugating the
drug to iRGD and the co-administration with Abraxane as the
drug. Both methods gave significantly higher anti-tumor activity
than the drug alone, and seemed equally effective in this regard
in the tumor system we studied (38). However, it should be noted
that the number of receptors at the target limits the efficacy of
the conjugated delivery. Calculations show that a gram of tumor
tissue is not likely to have more than a few picomoles of any given
receptor available for targeting of drugs with probes coupled to the
drug (1). Most drugs to be effective require greater concentrations
than could be delivered to this small an amount of receptor. The
co-administration mode does not have this limitation, as only the
triggering of the trans-tissue transport pathway is needed. Another
major advantage is that it is not necessary to conjugate the drug
to the homing peptide, which would create a new chemical entity
with the attendant regulatory hurdles.
LyP-1 coupled to Abraxane nanoparticles also increased the
efficacy of the drug (59) and iNGR promoted the activity of
FIGURE 2 | The tumor penetration cycle of CendR peptides. Following
systemic administration, tumor-penetrating peptides are initially recruited to
tumor blood vessels (2) followed by proteolytic processing to unmask the
CendR motif, and activation of NRP-1-binding (3, 4). NRP-1 engagement
triggers extravasation of the processed peptide and payload and triggers a
bulk transport process that increases delivery of payloads (6) and systemic
accessibility of blood-borne compounds, including unprocessesed
tumor-penetrating peptides for progressive penetration into tumor tissue (5). August 2013 | Volume 3 | Article 216 | 5
Page 5
Teesalu et al. Tumor-penetrating peptides
doxorubicin in a mouse tumor model in a way similar to iRGD
(48), by a factor of about 3. Importantly, the iRGD work with dox-
orubicin showed that there was no change in the main side effect of
this drug, cardiotoxicity. This side effect was nearly eliminated by a
threefold reduction of the drug dose. Thus, the tumor-penetrating
peptides can be used both to enhance the activity of anti-cancer
drugs, or lowering the side effect with the same anti-cancer activity,
or some of both.
The tumor-penetrating peptides can also enhance tumor imag-
ing, as demonstrated by coating iron oxide nanoparticles with
iRGD for MRI imaging. iRGD gave stronger images than a conven-
tional RGD peptide, CRGDC; the main difference was that iRGD
spread into the whole tumor, whereas only highlighted the tumor
vessels (13). LyP-1 has been used in optical imaging of tumors (11,
61) and atherosclerotic plaques (60), as well as in MRI and PET
imaging of plaques (61). LyP-1 homes to and penetrates into acti-
vated macrophages in tumors and atherosclerotic plaques (60, 61)
revealing a similarity between the macrophages in tumors and the
plaques (61). LyP-1 has also been shown to selectively accumulate
in tumor-draining lymph nodes prior to the arrival of tumor cells,
defining a premalignant niche in tumors (62).
The discovery of tumor-penetrating peptides has led to the iden-
tification of a new trans-tissue transport pathway, the C-end Rule
or CendR pathway. The physiological function of the CendR path-
way and its molecular workings are obviously important questions
to be answered in future studies. Activating the pathway in a
tumor-specific manner, which is accomplished with peptides the
CendR motif of which is activated in tumors, provides a way of
increasing the activity of anti-cancer drugs and enhancing tumor
imaging. Thus, the tumor-penetrating CendR peptides represent
a potentially significant advance in cancer treatment.
The authors’ original work reviewed in this article is supported
by Cancer Center Support Grant CA30199 to SBMRI, Innova-
tor Awards W81XWH-08-1-0727, W81XWH-09-0698 from the
Department of Defense, and grant CA CA152327 from the
National Cancer Institute. Tambet Teesalu is supported by Euro-
pean Research Council starting grant (GliomaDDS) and the
Wellcome Trust Award 095077/Z/10/Z.
The views and opinions of authors expressed on OER websites
do not necessarily state or reflect those of the U.S. Government,
and they may not be used for advertising or product endorsement
The opinions expressed herein are those of the author(s) and are
not necessarily representative of those of the Uniformed Services
University of the Health Sciences (USUHS), the Department of
Defense (DOD); or, the United States Army, Navy, or Air Force.
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Conflict of Interest Statement: Tambet
Teesalu, Kazuki N. Sugahara, and Erkki
Ruoslahti are shareholders in CendR
Therapeutics Inc., and Erkki Ruoslahti
is a shareholder in EnduRx Pharma-
ceuticals. The companies have rights to
some of the technology described in the
Received: 18 June 2013; paper pending
published: 27 June 2013; accepted: 06
August 2013; published online: 27 August
Citation: Teesalu T, Sugahara KN and
Ruoslahti E (2013) Tumor-penetrating
peptides. Front. Oncol. 3:216. doi:
This article was submitted to Pharmacol-
ogy of Anti-Cancer Drugs, a section of the
journal Frontiers in O ncology.
Copyright © 2013 Teesalu, Sugahara
and Ruoslahti. This is an open-access
article distributed under the terms of the
Creative Commons Attribution License
(CC BY). The use, distribution or
reproduction in othe r forums is permitted,
provided the original author(s) or licensor
are credited and that the original publica-
tion in this jour nal is c ited, in accordance
with accepted academic practice. No use,
distribution or reproduction is permit-
ted which does not comply with these
Frontiers in Oncology | Pharmacology of Anti-Cancer Drugs August 2013 | Volume 3 | Article 216 | 8
Page 8
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    • "If the tumor-cell killing motif is functional inside of the cells, the tumorhoming motif should satisfy both tumor specific targeting and penetration of cargo through the cell membranes of tumor cells. Although several studies have identified tumorhoming motifs that satisfy these conditions, tumorhoming motifs conjugated with a pro-apoptotic peptide, for example, D (KLAKLAK) 2 [21] as shown in Figure 3, or with a chemotherapeutic agent [27][28][29][30]have shown limited anti-tumor efficacy, possibly due to the poor killing activities of pro-apoptotic motifs, short circulating halflife , and/or tumor cell resistance. The serum stability of peptide-based drugs without modifications are, in general, less than 1 hour [31][32][33][34]. "
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    • "For protein-based therapeutics, targeting domain and therapeutic molecule are fused together as a recombinant protein with enhanced activity and tissue-specificity in conjugated delivery; (B,C) Bystander effect: Compounds co-injected with tissue-penetrating homing peptides are transported across the vessel wall and through tissue together with the peptides [12,42434445. No physical conjugation is needed between the targeting peptide and drug; the cell penetrating homing peptide " sweeps " co-injected drugs to its target (homing) tissue in tissue-specific fashion [12,42,45]. One of the benefits of using short peptides as targeting elements, is that the peptides are unlikely to be immunogenic in themselves because they are simply too small. "
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    • "In general, a well-thought-out prodrug strategy could achieve tissue-specific actions and reduce the undesired toxic effects, as the prodrug was designed according to the difference between the target environment and the abnormal physicochemical properties, like pH, temperature, over-expressed enzymes, receptors , and transporters etc. Considering that the common mechanism of the resistance to chemotherapy is the inability of drug transport across the cell membranes, transport moiety is especially critical in the case of anticancer therapy for protein drug [16], and various prodrug strategies were developed to achieve the selectivity issue161718192021. Except for Prodrug strategy, various approaches based on carriers, like antibodies [22], tumor homing peptides232425 , various nanoparti- cles262728293031, red blood cells313233, or even small molecule ligands [34, 35] have been utilized in the purpose of directing the macromolecular drugs only to the cancer cells [36,37]. Our strategy, therefore, combines both of the attributes of prodrug and target drug delivery methods into a single delivery system, expecting to deliver the macromolecular drugs to specific tissue targets with minimal toxicity. "
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