Medicine in Drug Discovery 10 (2021) 100092

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Medicine in Drug Discovery

journal homepage: www.elsevier.com/locate/medid

Research Paper

Cell-penetrating peptides as part of therapeutics used in cancer research

Martin Matijass, Ines Neundorf ⇑
University of Cologne, Department of Chemistry, Institute for Biochemistry, Zuelpicher Str. 47a, D-50674 Cologne, Germany

A R T I C L E I N F O A B S T R A C T

Keywords: Peptides have gained more and more interest as therapeutics for different fields of medical indications. Built up
Cell‐penetrating peptides via a peptide bond from naturally occurring amino acids they stand out by their high target selectivity and good
Peptide–drug conjugates biocompatibility. However, using chemical peptide synthesis also modifications are easily introduced and offer
Drug delivery various opportunities to fine‐tune their activity spectrum. One of the main areas of the global peptide market is
Peptide therapeutics
oncology and during the past years, many peptide therapeutics with anti‐tumor activity have been reported
Cancer therapy
that evoke metastasis in malignant cells or evasion of cell death. Cell‐penetrating peptides (CPPs) are a partic-
ular group of bioactive peptides that can be naturally or synthetically derived and have the ability to autono-
mously translocate in cells and transport various cargoes with them. These versatile carriers have been widely
used to deliver different kinds of therapeutic molecules, and have also found application in cancer research.
Within this review we will summarize recent efforts made in this direction with a focus on the specific cancer
indications for which CPP‐related therapeutics have been developed and studied.

1. Introduction development market. At present, three main areas are occupied by

Since several years, the global peptide therapeutics market size is
steadily increasing and is anticipated to rise over the coming years
[1]. Peptides offer a great opportunity in the creation of effective drugs
with their size being in between small‐molecule drugs and biological
compounds, e.g. proteins. For instance, peptides bind with high affin-
ity to their receptors, are usually accompanied by low immunogenic-
ity, and yield well‐tolerated metabolic building blocks after
proteolytic degradation. Despite their high biocompatibility, they
stand out by their ease of synthesis and the possibility to introduce var-
ious modifications. Above all, owing to their smaller size compared to
e.g. antibodies, peptides may by‐pass off‐target effects and thus, facil-
itate the administration of lower doses. Shortcomings that come along
with peptide therapeutics are, for instance, their relatively short
plasma half‐life and negligible oral bioavailability, reasons that limit
the general development progress in peptide therapeutic discovery.
However, as evolution in synthesis techniques is continuing, peptides
are more and more demanded and positively influence the drug
peptide therapeutics, and, besides metabolic indications and
endocrinology, about 17% of the approved peptide therapeutics target
indications related to oncology [2]. Cancer happens when cells divide
in an uncontrolled way, what is often the result of mutations, or abnor-
mal changes in the genes responsible for regulating the growth of cells,
and still, it is one of the main causes of death worldwide. Although
many chemotherapeutics with high potential are on the market, many
of them are adversely affected from insufficient pharmacological pro-
files. For example, while paclitaxel is one of the most applied drugs in
chemotherapy, it suffers from poor water‐solubility leading to a disap-
pointing pharmacokinetic performance. This is not the only case in
which chemotherapy causes unfavorable effects that, as a result, com-
promise the patient’s quality of life. Thus, one of the many efforts in
cancer research is to find new diagnostic and therapeutic strategies
that limit or even circumvent the mentioned disadvantages of conven-
tional drugs. Going in this direction, one way to increase the bioavail-
ability of small‐molecule medicals might be the use of efficient
peptidic drug carriers, so‐called cell‐penetrating peptides (CPPs).

Abbreviations: ACM, acute myeloid leukemia; ACPP, activatable cell‐penetrating peptide; ANG, Angiopep; ASO, antisense oligonucleotide; BBB, blood–brain‐barrier; CPPs, cell‐
penetrating peptides; CPT, camptothecin; CRC, colorectal cancer; Cur, curcumin; EGFP, enhanced green fluorescent protein; EPS8, epidermal growth factor receptor pathway substrate
no.8; GBM, glioblastoma; IA‐TCH, intra‐arterially‐transient cerebral hyperfusion; Mad, multiple antigen domain; MDM, murine double minute; MMP, matrix metalloproteinase; MTX,
methotrexan; NSCLC, non‐small cell lung cancer; NLS, nuclear localization sequence; NRP, neuropilin; PCNA, proliferating cell nuclear antigen; PEI, polyethyleneimine; PIP2,
phosphatidylinositol‐bisphosphate; PPI, protein–protein interaction; PS, phosphatidylserine; PTX, paclitaxel; RNP, ribonucleoprotein; SCLC, small cell lung cancer; STAT3, signal
transducer and activator of transcription 3; TLR, toll‐like receptor; VEGF, vascular endothelial growth factor.
⇑ Corresponding author.
E-mail address: ines.neundorf@uni-koeln.de (I. Neundorf).

https://doi.org/10.1016/j.medidd.2021.100092
Received 28 February 2021; Revised 5 April 2021; Accepted 6 April 2021
Available online 16 April 2021
2590-0986/© 2021 The Author(s). Published by Elsevier B.V.
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
M. Matijass, I. Neundorf
Within this work, we question if and how CPPs may contribute to
the specific niche of cancer diagnostics and therapeutics within the
global peptide drug market. In this regard, this review aims to update
recent progress made in this direction, emphasizing to summarize the
application of CPPs and CPP–drug conjugates in certain fields of can-
cer research. We are aware that excellent reviews exist within this field
[3–7] and will, therefore, focus on the very recent systems published
within the last years.
2. Cell-penetrating peptides – short overview
Cell‐penetrating peptides comprise a class of peptides that own the
potential to traverse the cell membrane and to act as carriers for cova-
lently or non‐covalently attached cargoes of different sizes, from small
molecules, to peptides, nucleic acids, proteins, or even particles. Often,
these are short sequences, e.g. in between 5 and 30 amino acids, that
have no need of a specific transport system or receptor for achieving
translocation. Since their first description in the 1980’s [8], an unclear
number of CPPs has been presented having different sequence lengths,
amino acid compositions and nowadays also attached/included modi-
fications. CPPs can be classified in different ways, in naturally or syn-
thetically derived, linear or cyclic compounds, or based on their
physicochemical characteristics into hydrophobic, hydrophilic or
amphipathic sequences. Meanwhile, it is also possible to predict their
features using suitable database tools [9]. One often observed struc-
tural property is the presence of basic amino acid residues, mainly
arginines, which are known to support the first contact of the peptides
with anionic components at the outer layer of the plasma membrane.
However, also amphipathic or fully hydrophobic CPPs are known. In
any case, CPP‐lipid interaction is an important key required for the
activity and translocation of CPPs, and often has a direct correlation
with the permeation efficiency across the lipid bilayer [10]. When
CPPs approach the plasma membrane, they interact with negatively
charged substituents of the lipid bilayer, for instance heparane sul-
fates, leading to structuring and accumulation of the peptides at the
lipid phase [11]. This clustering then initiates distinct events resulting
in the uptake of the CPP(‐cargo complex). In fact, this process of cellu-
lar uptake may follow very different routes and is dependent on vari-
ous more factors, as concentration, pH, temperature, CPP and cell‐lines
used, cargo attached, etc. Owing to this, the uptake mechanisms of
CPPs alone, as well as when in presence of their cargoes, are still not
really clear and a matter of intense discussion. However, two main
entry pathways are predicted for cellular uptake of CPPs. On the one
hand, they are able to directly permeate the plasma membrane follow-
ing mechanisms that were already proposed for other membrane‐
active peptides such as antimicrobial peptides [12]. Very recently, it
was hypothesized that for cationic CPPs positive membrane curvature
is another crucial prerequisite to promote such cellular uptake, and
that the use of chemical or physical stimuli may become a useful
way to control CPP invasion [13]. On the other hand, when CPPs
are fused to any cargo of larger size, they typically use different kinds
of energy‐dependent endocytosis to get access to the cell interior [14].
The nature of this way of internalization demands the effective escape
of the cargo out of the endosomes and thus, displays one of the limita-
tions that come with the application of CPPs. Many drugs have their
targets in the nuclei or cytosol and therefore, efficient endosomal out-
flow of the drug and further transport to its intracellular destination is
a real crucial point. During the last years, several exciting strategies
have been proposed to circumvent endocytic uptake or to support
endosomal release [15]. For instance, one very effective method for
cytosolic localization of CPPs is cyclization and meanwhile, many
cyclized peptide therapeutics including CPPs have been described
[16]. It has been postulated that small ring sizes, the presence of both
arginine and hydrophobic residues, as well as cyclization are critical
features to improve cellular uptake [17]. Also cyclic peptides enter
2
Medicine in Drug Discovery 10 (2021) 100092
cells by various different mechanisms, however, for many of them it
has been postulated that conformational transition significantly con-
tributes to membrane permeability [18]. Moreover, cyclic peptides
have greater resistance to proteolytic degradation, another important
fact leading to higher intracellular accumulation.
For more information concerning background, uptake mechanisms
and development progress in CPP research, the reader is referred to
informative reviews published very recently in this field [19–21].
3. CPPs in clinical trials to treat different kinds of cancer
Although many different CPPs have been discovered and studied,
the number of CPPs being part of a therapeutic or diagnostic investi-
gated in (pre)clinical trials is relatively small and often goes back to
the initially identified sequences Tat and penetratin [22,23], or the
synthetic poly‐arginines [24,25]. Probably, the reason for this phe-
nomenon is that researchers have gained a more comprehensive pic-
ture of the activity of these peptides, what helps to fine‐tune the
pharmacological profile of the future CPP‐based therapeutic. Interest-
ingly, this trend is not straightly followed when looking at the com-
pounds that are currently being investigated in clinical studies for
different kinds of cancer. Here, we observed more cell‐permeable pep-
tide therapeutics having an intrinsic anti‐tumor activity, e.g. either
related to the specific targeting of distinct protein–protein interactions
(PPIs), or by executing other mechanisms leading directly to cell
death. Anyway, as can be depicted from Table 1, CPP‐based com-
pounds are currently being studied for their utilization in breast can-
cer, colorectal cancer, lung cancer, leukemia, lymphoma and
metastatic, refractory or advanced solid tumors.
For example, PEP Therapy uses CPPs fused to bioactive peptides
interfering with PPIs, thus inhibiting pathological mechanisms. Beside
intracellular uptake of the interfering peptide mediated by the CPP,
this strategy promises high‐selectivity, stability and safety profile.
Their compound PEP‐010 is a proapoptotic agent developed to block
caspase‐9/PP2A interaction. Based on the promising preclinical data,
the company is currently initiating a Phase Ia/Ib study.
Another strategy is followed using the compound ATP128, which
was initially developed by AMAL Technology and is now evaluated
in combination with Boehringer‐Ingelheim’s anti PD‐1 antibody
BI754091 in a Phase Ib trial (‘KISIMA‐01’) [26]. This agent is a chi-
meric recombinant protein vaccine comprising a cell‐penetrating pep-
tide for antigen delivery, a TLR peptide agonist for self‐adjuvanticity
and a multiple antigenic domain (Mad) that can be tailored for any
specific indication. The current study is designed to treat colorectal
cancer.
The lead drug candidate of APIM Therapeutics is ATX‐101 and cur-
rently being tested in a phase I clinical study in advanced cancer
patients. ATX‐101 is a cell‐penetrating peptide including a cell pene-
tration and a nuclear localization sequence. It targets the proliferating
cell nuclear antigen (PCNA), which is an important scaffold protein
and involved in various cellular processes. Upon interaction with
PCNA, significant changes in gene expression and cellular metabolism
occur leading to apoptosis in cancer cells [27].
Aileron Therapeutics is currently underway with a phase II study
evaluating ALRN‐6924. This is a cell‐permeable stapled peptide target-
ing the PPI between the p53 tumor suppressor protein and its endoge-
nous inhibitors murine double minute X (MDMX) and 2 (MDM2) [28].
The clinical study is designed to target liposarcoma, osteosarcoma and
glioblastoma, but also other cancer types are efficiently targeted as
lung cancer and leukemia.
Directly acting cell‐permeable anti‐cancer peptides based on redox
proteins (the cupredoxins) are developed by CDG Therapeutics. They
have different peptide therapeutics in pipeline that target selectively
cancer cells and induce cell cycle arrest. One of their major targets
are metastatic, p53‐positive solid tumors in the brain [29]. Therefore,
M. Matijass, I. Neundorf
Table 1
Examples of CPP-related compounds currently under investigation in clinical trials.
Name Company Cargo
PEP-010 PEP-Therapy Interfering peptide
ATP128 Boehringer-Ingelheim TLR agonist and multiple
antigenic domain
ATX-101 APIM Therapeutics PCNA targeting peptide
ALRN-6924 Aileron Therapeutics – Lung cancer, Leukemia,
p28 CDG Therapeutics – Refractory solid tumors
AVB-620 Avelas Biosciences Fluorescent dye
they have successfully established their cell‐penetrating peptides to
bypass the blood–brain‐barrier (BBB). One of their lead compounds,
namely p28, was already studied in a phase I clinical trial in adult
patients with metastatic solid tumors expressing p53. Additionally,
the same agent was investigated in a phase I clinical study in pediatric
patients with recurrent or progressive CNS tumors. Both studies are
completed and demonstrated that p28 was well tolerated and that a
favorable therapeutic index was achieved.
Another promising compound that has finished a clinical phase II/
III study is presented by Avelas Biosciences, namely pegloprastide
(AVB‐620) [30]. This compound serves as imaging agent for breast
cancer diagnosis and is based on an activatable CPP equipped with a
fluorescent dye for ratiometric imaging. It is administered by intra-
venous injection approximately 3–20 h prior to surgery and has shown
great potential in detecting cancer in margin specimens in real time.
Furthermore, a first in‐human phase I study has demonstrated safety
and tolerability of pegloprastide to woman with primary, non‐
recurrent breast cancer undergoing surgery.
As already mentioned, there seems to be an ongoing trend to target
PPIs by designing either chimeric peptides that comprise the PPI tar-
geting part and the CPP, or to create PPI targeting peptides having
intrinsic cell‐permeability. Of course, the latter approach would be
more desirable, since it might yield a shorter peptide that is easier
and more cost‐effective to produce in large scale and to develop as a
medical. However, principally, all the presented studies confirm the
success of CPP‐based diagnostics and therapeutics utilized for indica-
tions in oncology. All compounds studied are characterized by their
good pharmacological profiles and give hope for first approved CPP‐
based drugs in the near future. On the other side, since only few com-
pounds have made it into clinical studies, there is still a need to
increase the number of potent CPP‐based compounds with anti‐
tumor activity or useful as tumor‐diagnostics.
4. Current research on CPP-based therapeutics with indications in
oncology
Owing to the promising data that now exist substantiating the suc-
cess of CPP‐related therapeutics, more efforts are on the way to find
new advanced systems that meet the high criteria of an effective
peptide‐drug suitable for cancer medicine. Particularly, for the most
common cancer types including lung, breast, and colorectal cancer,
also CPP‐related therapeutics are under development and investiga-
tion. Within this chapter we will highlight some of these recent studies
including novel CPPs with enhanced activities. Furthermore, we have
noticed that the development of multimodal platforms combining
CPPs with particles, drugs and targeting units play an increasingly
important role, and therefore, we have also picked out some examples
here.
Medicine in Drug Discovery 10 (2021) 100092
Cancer Types Function Status Identifier
Metastatic solid Caspase 9 inhibitor Phase Ia/Ib NCT04733027
tumor cancer
Colorectal cancer Induces cytotoxic Phase Ib NCT04046445
T-cell response
Advanced solid tumors VEGFR and EGFR Phase I n.a.
inhibition
MDM2/MDMX inhibition Phase I/II NCT02909972, NCT03725436,
lymphoma NCT04022876, NCT02264613,
NCT03654716
Inhibition cancer Phase I NCT01975116, NCT00914914
cell proliferation
Breast cancer Imaging Phase I, NCT02391194, NCT03113825
Phase II/III
4.1. Brain cancer
Glioblastoma (GBM) is a very aggressive tumor type with a median
survival around 15 months. One restriction in GBM therapy is the pres-
ence of the blood–brain‐barrier (BBB) that protects against circulating
toxins and pathogens but allows only nutrients to reach the brain.
However, recent investigations have shown promising results for
CPP drug‐conjugate formulations to cross the BBB and target tumor
cells in the brain. For instance, Joshi et al. investigated the perfor-
mance of Tat when either intra‐arterially (IA) or intravenously injected
in vivo in 9L brain tumor cells of tumor bearing mice [31]. They
observed a four‐fold increase of fluorescently labeled CPP when it
was IA applied and the experiment done under transient cerebral
hyperfusion (TCH) conditions. In summary, Tat not only demonstrated
the ability to efficiently cross the BBB using the IA‐TCH method, but
was also localized in the brain to higher extent compared to conven-
tional intravenous injections, a fact that might be used for anticancer
drug delivery in future experiments.
Tat was also used as CPP for the effective delivery of modified poly-
ethyleneimine (PEI) nanocomplexes loaded with angiogenesis‐
inhibiting secretory endostatin gene (pVAXI‐En) to treat glioma. For
selective targeting, a tandem peptide comprised of Tat and AT7 was
first constructed and evaluated. Generally, CPPs are taken up non‐
selectively in nearly all cell types, a fact that makes it difficult to create
a tumor‐selective therapeutic. Therefore, targeting moieties are often
additionally included in the design of a CPP‐based therapeutic. This
targeting moiety should direct the compound to the respective tumor
tissue/cells, while the CPP may be responsible for intracellular uptake
of the drug at the target site. We have recently successfully designed
such a cancer‐targeting CPP‐based drug delivery platform [32]. How-
ever, AT7 is a peptide recognizing the vascular endothelial growth fac-
tor receptor 2 (VEGFR‐2) and neuropilin‐1 (NRP‐1) receptor both of
which are overexpressed on new blood vessels in glioma and thus, rep-
resent valuable targets. Indeed, the final nanoparticle construct signif-
icantly suppressed the tube formation and migration of endothelial
cells, and demonstrated high activity in reducing the microvasculature
in orthotopic U87 glioma‐bearing nude mice [33].
Another novel GBM therapy, but also including Tat as CPP, targets
dysregulated phosphoinositide signaling. Therefore, Eustace et al. used
peptide mimetics derived from the phospholipid binding domain of
myristoylated alanine‐rich C‐kinase substrate (MARCKS) and fused
them to Tat to make them available for the brain [34]. Their in vitro
and in vivo results impressively illustrated how these cell‐permeable
MARCKS peptides induced rapid, potent, and selective cytolytic fea-
tures against GBM, and provide new opportunities to improve patients
outcomes.
In an effort to improve brain delivery for chemotherapeutics that
target mitochondrial potassium channel mtKv1.3 and selectively
3
M. Matijass, I. Neundorf
induce oxidative stress, Parrasia et al. coupled a 5‐(4‐phenoxybutoxy)
psoralen‐derived compound (PAPTP) to the CPP Tat or Angiopep‐2,
respectively [35]. Both constructs demonstrated efficient delivery of
the construct in the brain after intravenous injection to mice and there-
fore, open up new avenues for the further development of these
peptide‐drug conjugates for brain cancer.
In past decades, great progress has been made in the development
of drugs based on RNA interference (RNAi), playing also an important
role for cancer gene therapy. However, one main limitation in siRNA‐
based drugs is still the safe delivery to their site of action. Srimanee
et al. investigated the potential of CPPs PF14 and PF28 to deliver
siRNA into human glioblastoma cells U87. Successful cellular uptake
of siRNA was confirmed by silencing luciferase activity in the glioblas-
toma cells. For a specific tumor targeting, the amphipathic CPPs were
both covalently or non‐covalently conjugated to a modified glioblas-
toma homing peptide Angiopep (ANG). PF14:siRNA in complex with
the homing peptide showed promising results in gene silencing, as this
construct was able to reach 80% knockdown of luciferase expression.
Comparing the uptake of PF14:siRNA between U87 and non‐glioma
cells, selectivity towards the cancer cells was observed. However,
crossing the BBB in vivo has not been investigated until now [36].
4.2. Breast cancer
Breast cancer is the most common cancer type affecting women,
and the risk factors can be age, family history and life style factors
(e.g. smoking, obesity). Invasive breast cancer is the more common
type of this cancer, while triple‐negative breast cancer is a more
uncommon type. The survival rate for breast cancer is generally good,
particularly, if the diagnosis comes at an early stage. However, since
chemotherapy is also one main treatment option for this cancer type,
still more active and selective drugs affecting only breast cancer cells
are developed.
In this respect, Mitra and colleagues designed an all hydrocarbon
stapled α‐helical peptide in order to bind to a protein named RAB25,
which is involved in the pathogenesis of various cancers including
breast cancer [37]. As complexation of RAB25 with RAB‐family inter-
action proteins (FIP) has been shown to execute dysregulated signal-
ing, the constructed peptide is generated to bind RAB25 in order to
functionally block formation of a RAB25:FIP complex and to inhibit
the phenotype associated with RAB25. Indeed, inhibition of cell migra-
tion as well as reduced proliferation of MCF‐7 breast cancer cells was
observed when cells were transfected with the designed peptides, indi-
cating that disrupting RAB25 binding might serve as a new point of
intervening on breast cancer cell migration.
One year later, Tansi et al. studied several different CPPs, and one
of it was termed mini Crotamine, which was initially derived from a
venom and comprised the following sequence: YKQSHKKGGKKGSG.
Fluorescently labeled peptides were investigated in several cancer cell
lines, whereby the fluorophore was either non‐covalently or covalently
attached. Interestingly, highest cellular uptake was found particularly
for this mini Crotamine peptide when using breast cancer cells, com-
pared to other cancer and non‐cancerous cell lines tested. Therefore,
the authors concluded that this CPP might be used for further studies
on the identification of heterogenic cancer subpopulations, and that it
could possibly advance to a tool for future theranostic image‐guided
applications [38].
Based on the already mentioned peptide PF14, Künnapuu and col-
leagues designed a matrix metalloproteinases (MMP)‐2/‐9 activatable
cell‐penetrating peptide (ACPP) PF144 [39]. The cell‐penetrating moi-
ety of the peptides were masked with polyethylene glycol (PEG) mole-
cules at the C‐terminal end of the peptides via a MMP‐2/‐9 cleavable
linker. This formulation aimed to prevent the decreased cellular
uptake into cancer cells shown before by PEGylated peptides. The
authors generated CPP/pDNA nanoparticles with pDNA expressing
short hairpin RNA for silencing vascular endothelial growth factor
4
Medicine in Drug Discovery 10 (2021) 100092
(shVEGF), as VEGF is highly overexpressed in tumor cells. Regulation
of VEGF protein levels is supposed to inhibit the formation of new
blood vessels and consequently leads to increased permeability of
tumor cells making them more accessible for anticancer agents. In
comparison with administration of the pDNA alone, delivery of this
nanocomplex into 4T1 breast cancer cells expressing MMPs demon-
strated efficient reduction in tumor growth in tumor bearing mice.
The effect of the transfected plasmid was shown to be active for about
a week. Additionally, no reduction of tumor growth was seen by mice
that were treated with the uncleavable peptides, indicating the effec-
tiveness of the ACPP [39].
In order to retain the advantages of CPPs while increasing their sta-
bility, El Sayed et al. used the cyclic CPP [W(WR)4K(βAla)] which they
coupled covalently via an amide linkage with either the anticancer
drug paclitaxel to form [W(WR)4K(βAla‐hemigluterate‐2‐O‐PTX)] or
modified camptothecin to form [W(WR)4K(βAla)‐S‐S‐CPT)]. Human
breast cancer cells MCF‐7 were incubated with these peptide drug con-
jugates. In particular, promising results were observed for the CPT con-
struct, where inhibition of cell proliferation of up to 62% was observed
[40].
A more sophisticated approach was presented when Mn:ZnS
nanoparticles were coated with different CPPs (namely pVec, pene-
tratin and nona‐arginine (R9)) and additionally loaded with paclitaxel
[41]. Inorganic Mn:ZnS nanoparticles have been widely used as imag-
ing agents owing to their beneficial cytocompatibility. Interestingly,
the developed construct demonstrated promising in vivo activity in
breast cancer xenograft models compared to PTX alone. In more detail,
maximum tumor localization and enhanced anti‐tumor efficacy was
obtained when R9 coated nanoparticles were used and thus, those
were referred to stand out as potential new theranostic nanocarriers
for PTX.
4.3. Lung cancer
Two main types of lung cancer exist, non‐small cell lung cancer
(NSCLC) and small cell lung cancer (SCLC), respectively, and they
are treated very differently. However, both treatment strategies
include surgery, radiation therapy, chemotherapy and immune
therapy.
In an effort to establish the CRISPR/Cas9 approach for application
in cancer therapy, Lostalé‐Seijo et al. investigated a strategy for trans-
porting the Cas9 ribonucleoprotein (RNP) into cancer cells. Therefore,
they designed a cell‐permeable peptide named PT24, constituting of an
amphiphilic peptide connected to a hydrophobic aldehyde tail via a
hydrazone bond [42]. Incubation with the Cas9 RNP led to the forma-
tion of supramolecular nanoparticles through noncovalent binding of
Cas9 RNP to PT24, able to deliver the cargo into the cell. Acceptable
efficacy was observed by the delivery of the ribonucleoprotein into
the lung cancer cell line A549, making this approach to a conceptually
new system in spite of the direct delivery of endonucleases.
Yang and colleagues generated selective cell‐penetrating peptide
(RLWMRWYSPRTRAYGC)‐functionalized polymersomes (SCPP‐PS),
which were constructed by the co‐self‐assembly of PEG‐P(TMC‐DTC)‐
PEI and SCPP‐PEG‐P(TMC‐DTC) copolymers. Those nanoparticles
were then loaded with the anticancer drug methotrexat disodium
(MTX) in order to test the capability of drug delivery. The nanocom-
plex transport system exhibited tumor suppression in A549 cells.
Moreover, in vivo studies in A549 tumor bearing mice demonstrated
increased survival rate of MTX‐SCPP‐PS compared to MTX stand alone
and MTX‐PS [43].
The transcription factor MYC is often upregulated in cancers and
correlates with high aggressiveness. Although there is a fear of side
effects, targeting MYC could be an opportunity in therapeutic cancer
research. In this context, Beaulieu et al. obtained promising results
with their purified Omomyc mini‐protein. They demonstrated efficient
cell‐penetration of Omomyc into A549 lung cancer cells. Moreover,
M. Matijass, I. Neundorf
mouse models with lung adenocarcinoma showed decreased tumor
progression, resulting in increased survival after systemic administra-
tion of the Omomyc mini‐protein and combination with a microtubule
targeting agent [44].
Another delivery system, namely recombinant lacaptin (RL2), was
used by Chinak et al. for the intracellular transport of nucleic acids.
RL2 was able to form non‐covalent complexes with pEGFP and anti
EGFP siRNA. Fluorescence microscopy of A549 cells transfected with
RL2:pEGFP confirmed the penetration of lacaptin into the cells and
the delivery of the plasmid DNA into the cells. In addition, also siRNA
delivery was successful which could be confirmed by co‐transfection of
RL2:pEGFP and RL2:siRNA [45].
To overcome the drawbacks of direct administration of curcumin
(Cur), Guo et al. developed an enzyme triggered CPP‐mediated
nanoparticle (MePEG‐Peptide‐Tri‐CL) platform to enable the delivery
of Cur. The generated construct demonstrated efficient anti‐
proliferation effects against A549 lung cancer cells confirming the
intracellular delivery of the cargo. Due to the fluorescence ability of
Cur, an additional confirmation of cellular uptake was observed. More-
over, by stimulating the environment in blood stability of this complex
was confirmed in vitro. Following in vivo studies in tumor bearing mice
demonstrated tumor growth inhibition of up to 77% [46].
4.4. Colorectal cancer
Colorectal cancer (CRC) is the second most deadly cancer type
worldwide. This term combines colon cancer beginning in the large
intestine (colon) and rectal cancer, which begins in the rectum. Treat-
ment strategies for CRC usually include combination of surgery, radi-
ation therapy and chemotherapy. Since many chemotherapeutics used
exhibit unwanted side effects, also in this case, novel treatment strate-
gies and diagnostic tools are searched for. In this respect, plant‐derived
peptides that exhibit anti‐tumor activity in CRC have been extensively
reported and might be an interesting opportunity [47]. However, con-
cerning CPP‐based therapeutics, recently, Zeng and colleagues
described an imaging probe for the early detection of CRC. Herein,
they designed an activatable cell‐penetrating peptide (ACPP), which
is activated by cleavage of MMP‐2 or ‐9, which are both overexpressed
on colorectal cancer cells. In addition, a fluorescent dye, namely Cy5,
was attached to the ACPP and the uptake of this construct into colorec-
tal cancer cells bearing mice was investigated. Significant higher fluo-
rescence intensities within the colorectal tumor cells compared to the
intensities of other organs were obtained, giving hope that this con-
struct could assist in early colorectal tumor localization [48].
Recently, a low molecular weight PEI derived delivery system was
established including an integrin targeting unit (cyclic RGD), a cell‐
penetrating peptide (nona‐arginine), as well as polyethylene oxide
and cholesterol to further improve the physicochemical properties of
the construct. It was demonstrated by the authors that these nanopar-
ticles could efficiently deliver siRNA targeting the signal transducer
and activator of transcription 3 (STAT3) into different colon cancer
models and induce strong anti‐cancer effects [49]. Moreover, in vivo
compatibility and activity was proven when the complex was adminis-
tered intravenously and suppressed lung metastasis tumor progression.
Thus, this vector system could be a real new alternative for siRNA, but
also for other RNA or even DNA therapeutic molecules.
4.5. Leukemia
Leukemia is a cancer of the blood cells and generally refers to can-
cers of the white blood cells. In fact, the direct causes of leukemia are
not known, but several factors have been identified that may increase
the risk, such as smoking, genetic disorders or blood disorders, to
name only some.
Epidermal growth factor receptor pathway substrate no.8 (EPS8)
occurs in various cancers and was previously described to be another
5
Medicine in Drug Discovery 10 (2021) 100092
opportunity in cancer targeting. In this context, Chen et al. developed
an anti‐acute myeloid leukemia (AML) peptide to target EPS8 in AML
cells. The peptide comprises TAT for facilitated penetration and a
nuclear localization signal (NLS) mimicking the NLS of EPS8. The
novel construct demonstrated reduced cell proliferation as well as cell
viability in AML cells. Administration of the peptide induced inhibi-
tion of tumor progression in mice bearing AML cells [50].
Another approach was used by Shah et al. who constructed a chi-
meric peptide, containing TAT as the CPP and an ASHL2 derived pep-
tide sequence, in order to bind DPY30, a chromatin modulator which
is highly upregulated in AML cancer cells. Binding of the ASHL2
domain to DPY30 is responsible for the methylation activity of the lat-
ter, however, direct correlation of the role in tumorigenesis had yet to
be investigated. Penetration of the peptides, inhibition of cell growth
of AML cell lines, as well as induced apoptosis was observed by incu-
bation of the generated peptide, which not only demonstrated the abil-
ity in targeting leukemia cells, but also confirmed that interfering the
DPY30/ASHL2 interaction might be a promising therapeutic approach
in future studies [51].
Banozci et al. designed an octa‐arginine (Arg8) CPP conjugated
with vindoline derivates. The latter are thought to destabilize the
microtubule network, however, initial studies administering these
alkaloids alone did not yield any anti‐tumor activities. The authors,
therefore, chemically transformed the vindoline derivatives and conju-
gated them with Arg8. This formulation displayed anti‐tumor activities
against human leukemia cells in preliminary in vivo studies [52].
4.6. Metastasis
One major problem in cancer medicine is the occurrence of a sec-
ondary or metastatic tumor. In fact, metastasis is one of the hallmarks
of cancer, since almost all types of cancer are capable to form metas-
tases. Therefore, strategies that inhibit metastasis are an important
focus of cancer research.
In this respect, Hu et al. generated a cell‐penetrating cabazitaxel
nanovehicel (r9‐CN), consisting of DSPE‐PEG2000 as the nanocarrier
material and a D‐version of poly‐arginine (r9) as the CPP unit [53]. Sig-
nificant inhibition of the viability of metastatic cancer cells was
demonstrated by the nano‐construct indicating efficient delivery of
the cabazitaxel cargo into the cells. Also, deep penetration in lymph
metastasis was observed resulting in inhibition of cell growth. In addi-
tion, administration of r9‐CN led to 90% reduction of distant lung can-
cer metastasis. Their obtained results were very promising for
counteracting metastasis.
In order to prevent cancer metastasis in mouse models, Gao et al.
investigated another CPP modification strategy. Here, palladium
nanosheets were used and coated with modified Tat‐peptides (intro-
duction of a CGG sequence at the N‐terminus) as well as with thiol‐
terminated mono‐methoxy PEG. The generated Pd‐Tat was then
administered in vivo to mice bearing 4T1 tumor metastasis. In a way
of combinational therapy, the mice were exposed to mild laser irradi-
ation below the permitted value. This combination consequently led to
a significantly enhanced inhibitory effect on cancer metastasis, owing
to an increased permeability of the nuclear membranes after
irradiation, and facilitated the delivery of Pd‐Tat. Either way, this ther-
apy demonstrated encouraging anti‐metastasis activity [54].
In another study, Niu et al. constructed a gold nanoparticle conju-
gated with the cell‐penetrating peptide Tat (AuPT). These nanoparti-
cles then were used to deliver pDNA coding for miRNA‐221
inhibitor gene into mice bearing melanoma cells. Overcoming the skin
barrier has been shown to be challenging for pDNA delivery. However,
topical application of this construct demonstrated prevention of metas-
tasis and progression of advanced melanoma, while cytotoxicity
towards non‐cancer cells was rather low. The authors then investi-
gated the tumor tissues after application of AuPT/mi221 and observed
apoptosis and necrosis of tumor cells. These findings demonstrated
M. Matijass, I. Neundorf
promising results in the therapy of skin cancer and prevention of
metastasis [55].
4.7. CPP-based formulations targeting various cancer indications
There are several works underway presenting conceptually new
ideas that might be used for cancer treatments in a more general
way, e.g. to find delivery systems or treatment strategies that can be
applied to different kinds of cancers.
Just recently, Xu et al. presented a peptide‐equipped exosome plat-
form useful for the delivery of antisense oligonucleotides (ASOs). In
this study, polyarginine was conjugated on the surface of HepG2 cells
derived exosomes via an amide bond [56]. The ASOs were either
directly inserted into the membrane of the exosomes, or interacted
by electrostatic forces with the cationic polyarginine sequences at
the surface of the exosomes. Thus, this platform offered a simplified
loading process without complicated genetic modification of producer
cells. The created compounds were shown to internalize in several dif-
ferent cancer cell lines. Moreover, they were tested with the therapeu-
tic ASO G3139, silencing successfully antiapoptotic Bcl‐2 in HepG2
cells. Since the exosomes not only served as delivery platform of the
ASO, but also supported the stability of the CPP, they represent a
promising new design concept for novel drug delivery systems for dif-
ferent cancer types.
Also dedicated to the design of non‐viral gene delivery vectors was
the recent study of McErlean et al. [57] Herein, a 15 amino acid linear
peptide, termed CHAT, was presented that was rationally optimized in
its sequence for facilitating nucleic acid complexation, cellular uptake,
as well as endosomal release. Functional plasmid DNA was complexed
with the peptides to produce nanocomplexes, which were efficiently
taken up by breast and prostate cancer cells and in addition, elicited
reporter‐gene expression in vivo after intra‐tumoral and intravenous
delivery. Thus, this new delivery vector might be applied in a general
way for effective intracellular pDNA transport.
On the other side, it has been found out, when using CPPs for the
generation of chimeric peptides that the CPP unit used has to be care-
fully investigated for its cellular uptake mechanism and cytotoxic
properties. To overcome the limited uptake of peptides targeting PPIs,
Philippe et al. conjugated KD3, a non‐cell permeable but potent and
specific peptide inhibitor of p53:MDM2 and p53:MDMX interactions,
with a different set of cyclic cell‐penetrating peptides. The resulting
conjugates were tested against several different cancer cell lines and
it was highlighted that the attached CPPs helped to translocate the
high affinity peptide binders, but that the obtained cell death mecha-
nism were dependent of the CPP attached. So, to make fine‐tuning of
the system possible, a deeper analysis of the CPP characteristics in the
specifically used system would be required [16].
Another interesting new way of anticancer therapy was recently
presented by Serulla et al. The authors designed Tat‐RasGAO317‐326,
a CPP‐based compound having both anticancer and antimicrobial
activity [58]. Notably, this peptide was able to lyse cancer cells by tar-
geting specific plasma membrane lipids, such as phosphatidylinositol‐
bisphosphate (PIP2) and phosphatidylserine (PS). It induced a
necrotic‐like form of death and thus, represents a real alternative to
common anticancer agents. Additionally, it might circumvent the
problems with resistance‐building often occurring for drugs regulating
cell death pathways.
We have recently generated so called‐ CaaX‐peptides comprising a
cell‐penetrating peptide, termed sC18*, and a C‐terminal CaaX motif
[59]. We recognized high cellular uptake of those peptides and signif-
icant cytotoxicity when applied to different cancer cell lines, while
non‐cancerous cells remained unharmed. Further results let us hypoth-
esize that those novel peptides get selectively enriched intracellularly
upon prenylation at the cysteine of the CaaX‐motif, and that they were
able to interfere with KRas‐related signaling pathways, probably lead-
ing to the observed cell death in cancer cells. Thus, this new approach
6
Medicine in Drug Discovery 10 (2021) 100092
of processing and interfering with post‐translational modification
might open up a new field for the development of drugs targeting
prenylated proteins relevant in cancer.
5. Conclusion
The studies summarized herein have shown that CPPs can be devel-
oped in two ways for oncology research, e.g. as drug transporters, or as
therapeutic molecules having intrinsic anti‐cancer activity. Since sev-
eral years, coupling of a bioactive molecule to a CPP, yielding a
peptide‐drug conjugate, has been successfully established to improve
its pharmacologic properties. And by enabling efficient delivery of
the drug to its intracellular target site, exciting results for cancer diag-
nosis and therapy have been obtained. Beyond that, recent years have
revealed a great interest in targeting protein–protein interactions by
small peptides, and researchers have pushed forces in equipping such
peptides also with cell‐permeability. The design of such therapeutics
would be innovative and smart and would circumvent the need of
additional transporters for cellular uptake.
However, although several CPP‐related compounds are in clinical
trials for diagnosis and therapy of cancer, until now, none is FDA
approved. Therefore, present research on CPP‐based therapeutics is
devoted to solve issues that come with the utilization of CPPs, such
as low stability or lack of cell selectivity. In our opinion, both issues
may be solved by the use of multimodal systems constructed for
instance by taking nanoparticles as platforms that can be equipped
with a number of different functional molecules, such as a drug, target-
ing unit and CPP. Indeed, several studies are underway to find versa-
tile synthesis strategies and to analyze the impact of such systems.
Future will show which direction the whole field will take, what-
ever it will be, we are sure that it will bring new exciting molecules
with high potency useful for cancer medicine.
CRediT authorship contribution statement
Martin Matijass: Conceptualization, Data curation, Writing ‐ orig-
inal draft. Ines Neundorf: Conceptualization, Data curation, Writing ‐
review & editing, Funding acquisition, Project administration,
Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgements
I.N. highly appreciates the great support by M.M. to write and final-
ize this work.
Funding Sources
This work was supported by the German Research Foundation
(grant number NE 1419/10‐1).
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