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`Protein&Cell
`
`Protein Cell 2019, 10(11):787–807
`https://doi.org/10.1007/s13238-019-0639-7
`
`Protein & Cell
`
`REVIEW
`Phage display screening of therapeutic
`peptide for cancer targeting and therapy
`
`Phei Er Saw1, Er-Wei Song1,2&
`
`1 Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Sun Yat-sen Memorial
`Hospital, Sun Yat-sen University, Guangzhou 510120, China
`2 Breast Tumor Center, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China
`& Correspondence: songew@mail.sysu.edu.cn (E.-W. Song)
`Received November 30, 2018 Accepted April 21, 2019
`
`ABSTRACT
`
`been
`has
`technology
`display
`phage
`Recently,
`announced as the recipient of Nobel Prize in Chemistry
`2018. Phage display technique allows high affinity tar-
`get-binding peptides to be selected from a complex
`mixture pool of billions of displayed peptides on phage
`in a combinatorial library and could be further enriched
`through the biopanning process; proving to be a pow-
`erful technique in the screening of peptide with high
`affinity and selectivity. In this review, we will first dis-
`cuss the modifications in phage display techniques
`used to isolate various cancer-specific ligands by
`in situ, in vitro, in vivo, and ex vivo screening methods.
`We will then discuss prominent examples of solid tumor
`targeting-peptides; namely peptide targeting tumor
`vasculature, tumor microenvironment (TME) and over-
`expressed receptors on cancer cells identified through
`phage display screening. We will also discuss the cur-
`rent challenges and future outlook for targeting peptide-
`based therapeutics in the clinics.
`
`KEYWORDS phage display, tumor targeting peptide,
`tumor vasculature, tumor microenvironment, tumor stromal
`cells, over-expressed receptor
`
`INTRODUCTION
`
`Peptides are 2-dimensional, linear chains of amino acids,
`which are usually short (less than 50 AA) in length (Hayashi
`et al., 2012). They are either designed by rational computing
`methods or phage display screening to obtain peptides that
`binds with high specificity to the target of interest, with a
`possibility of modulating the target (Marqus et al., 2017).
`Compared to antibodies (∼150 kDa), peptides are relatively
`
`© The Author(s) 2019
`
`small (∼3–5 kDa) and therefore easy to synthesize and
`modified (Boohaker et al., 2012), have higher cell membrane
`penetration, and possess less immunogenicity. In cancer
`therapy, these peptides can be used as a targeting ligand
`assisting specific delivery of cytotoxic drug specifically into
`the tumor vasculature, tumor microenvironment or into the
`cancer cells. On the other hand, peptides could also be
`delivered intracellularly to target cancer specific upregulated
`transcription factors, oncogenes or enzymes (Jyothi, 2012;
`Marqus et al., 2017). The general comparison between
`antibody and peptide are summarized in Table 1.
`Herein, we will review the utilization of phage display
`biopanning with modifications gearing towards in situ,
`in vitro, in vivo, ex vivo and in human application for high
`affinity peptide screening. We will also provide a compre-
`hensive discussion on the latest discovery of tumor target-
`ing-peptides; namely the peptides targeting (1)
`tumor
`vasculature,
`(2)
`tumor microenvironment
`(TME) and (3)
`over-expressed receptors on cancer cells.
`
`Phage display technology and biopanning strategies
`
`In 1985, George Smith first described phage display by
`demonstrating the ability of a filamentous phage to display
`peptide by fusing the library of peptide sequence into the
`virus’s capsid protein (Smith, 1985). Since the peptide was
`displayed on the viral surface, selection could be done to
`isolate those with the highest binding affinity towards a tar-
`get. In the same year, Geroge Pieczenik filed a patent also
`describing the generation of phage display libraries in detail
`(US patent, 5866363). However,
`the application of
`this
`technology was pioneered by Greg Winter and his col-
`leagues at
`the Scripps Research Institute for display of
`proteins (specifically antibodies)
`for
`therapeutic protein
`engineering. Due to their contribution in phage display
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`were constructed via cloning of combinatorial DNA
`sequence (Fig. 2A). This library will be amplified prior to
`biopanning (Fig. 2B). The second step is the “target cap-
`turing step”, in which the phage library is incubated with
`target molecule for a specific time to allow binding (Fig. 2C).
`The third step is to “remove unbound & nonspecific phages”
`by using repetitive washing to remove any unbound and
`non-target specific phages (Fig. 2D). The fourth step is the
`“elution step”, in which target-bound phages are separated
`after a short incubation with low pH buffer or by competitive
`elution (Fig. 2E). Finally, in the fifth step “infection stage”, the
`eluted phages are infected in bacteria to amplify selected
`phages, making a new and more selective phage library that
`should be applied in a next round of biopanning (Fig. 2F and
`2G).
`In general, three to five rounds of biopanning are neces-
`sary to isolate specific and high affinity peptide binders.
`Nonspecific phages are removed and phages with high
`affinity for the target are isolated by increasing the stringency
`in each round of biopanning by increasing the number of
`washing and decreasing the amount of target molecule. At
`the end of biopanning, phage ELISA and DNA sequencing
`are used for identification of individually specific phage with
`high affinity to target.
`
`Ample research to isolate high affinity peptide by phage
`display screening
`
`Although in situ phage display screening using immobilized
`antigen is capable of generating high affinity and specificity
`peptide (Kim et al., 2012b), to better mimic cellular and body
`condition, ample researches are being done on in vitro,
`in vivo (Liu et al., 2018), ex vivo (Sorensen and Kristensen,
`2011) and even in cancer patient
`(Krag et al., 2006)
`screening for high affinity peptide in a heterogenous envi-
`ronment as this is a closer representation to their original
`condition.
`
`Homogenous in situ screening
`
`Homogenous in situ screening requires only the specific
`target to be coated on a 96-well (Fig. 3A). A single target
`exposure guarantees the isolation of target-specific peptide,
`without external interference from non-specific binding. This
`method is also the easiest, as all experiments could be
`carried out without living system (i.e., cell culture, animal
`model, patient samples). The disadvantages of
`in situ
`screening includes the risk of non-specific binding of the
`isolated peptide when exposed to in vitro or in vivo system.
`In addition, the target is artificially coated onto the plate,
`which could be misrepresent the actual secondary structure
`of the target in a living system, therefore increases the risk of
`isolating a peptide that only binds to the receptor in this
`particular setting (Kim et al., 2012b).
`
`Table 1. The advantages of peptide as compared to antibody
`
`Antibody
`
`150 kDa
`pmol/L–nmol/L
`
`Peptide
`3–5 kDa
`pmol/L–nmol/L
`
`Size
`Affinity (KD)
`Immune response
`
`Tissue penetration
`
`Intracellular target
`
`Research cost
`
`Production cost
`
`Developing speed
`
`Little
`
`Low
`
`No
`
`High
`
`High
`Months–years
`
`Little
`
`High
`
`Yes
`
`Low
`
`Low
`
`Months
`
`Minimal
`
`Patent barriers
`
`High
`
`technique development and the enormous implication of
`phage display technology, Smith and Winter were both
`awarded a quarter share of the 2018 Nobel Prize in chem-
`istry, while the other half was awarded to Frances Arnold.
`Phage-display is a powerful technology for screening and
`isolating target specific peptides. This method utilizes bac-
`teriophage to display foreign peptides or antibodies on their
`surface through insertion of the gene encoding the corre-
`sponding polypeptides into the phage genome. For display
`of foreign polypeptides on the bacteriophage, the desired
`DNA sequence is inserted into the M13 phage pIII or pVIII
`gene (Fig. 1). The methodology using the major coat protein
`pVIII provides a multivalent display, however only short
`peptides (6–7 AA) could be displayed on pVIII gene.
`Therefore, most combinatorial libraries such as antibodies or
`proteins have been displayed using minor coat pIII. Since
`there could be only 3–5 copies of pIII protein per phage, this
`method limits the copy number but the length of foreign or
`synthetic polypeptides that can be expressed (Fig. 1).
`The phage selection method, referred to as biopanning, is
`an affinity selection process that
`isolates target-binding
`molecules. As explained in Fig. 2, generally phage display
`based biopanning consists of five screening steps for
`selection of peptides. The first step is “library construction &
`amplification” where polypeptide-displayed phage libraries
`
`5 nm
`
`M13 bacteriophage
`
`1,000 nm
`
`pVII + pIX
`pVIII
`pIII
`Figure 1. A typical representation of M13 phage with about
`1,000 nm in length and 5 nm wide. The major coat proteins are
`pIII (green), pVIII (purple) and pVII + pIX complex (yellow + red).
`
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`Protein&Cell
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`Tumor targeting peptide for cancer therapy
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`REVIEW
`
`A
`
`Library construction and amplification
`
`G
`
`Pick colonies and grow
`large quantity of phages
`F
`
`B
`
`E
`
`Target capturing
`
`C
`
`D
`
`Infect into bacteria and
`grow colonies on plate
`
`Elution of target-
`specific phages
`
`Co-incubation of
`phage and target
`
`Remove unbound
`& nonspecific phages
`
`Figure 2. The general scheme of phage display technique and biopanning selection of high affinity peptide. Peptide-based
`library is first obtained either commercially or specifically designed to cater for specific needs of each experiment.
`
`In vitro cell screening
`
`In vitro cell screening offers high-throughput approach for
`identifying multiple peptides that bind specifically to a single
`cell (i.e., cell lines or primary cells) and can be performed on
`adherent cells (live or fixed) (Fig. 3B). Advantages of using
`whole cell approach includes retaining their biological func-
`tions and activities, proper folding, 3-dimensional structure,
`receptor expression level and their association with neigh-
`boring proteins. Modified selection protocols could be used
`to isolate internalized peptides.
`Importantly,
`in vitro cell
`biopanning could identify novel cell surface receptors with
`unknown biological functions, which could be used to pro-
`vide information on specific molecular changes (i.e.,
`expression level of certain protein and their localization in
`normal vs. cancer cells) (Arap et al., 2002b; Zhao et al.,
`2007; Sun et al., 2012; Wu et al., 2016).
`
`In vivo screening
`
`By performing biopanning and selection in a living animal,
`organ-specific peptides could be isolated (Fig. 3C). Roush-
`lati and co-workers first described in vivo phage display
`technology in 1996 (Pasqualini and Ruoslahti, 1996). For
`in vivo biopanning protocol,
`the biopanning selection is
`similar to that of the in vitro screening, the difference being
`the peptide phage library was introduced into the animal via
`systemic intravenous injection and allowed binding to occur
`within 1–2 h (as peptide-displayed phage is estimated to
`bound to target within 5–15 min (Laakkonen et al., 2002; Lee
`et al., 2007; Lo et al., 2008)), after which the animals will be
`perfused to remove unbound phages, sacrificed, and the
`desired organs will be collected and homogenized. Tissue-
`specific phage should increase after 3–5 rounds of biopan-
`ning (Rajotte et al., 1998; Lee et al., 2007; Chang et al.,
`
`2009). Through this approach, various types of tumor and
`malignant tissue vasculature have been identified (i.e., RGD-
`4C, NGR and GSL peptide (Koivunen et al., 1995; Pas-
`qualini et al., 1997; Ruoslahti, 2000); detailed explanation
`below). One of the major pitfalls in using in vivo phage dis-
`play technology is that the peptides may not be translated
`into human due to the differences of peptide binding
`between species (Wu et al., 2016).
`
`Ex vivo screening
`
`This method, first published in Nature in 2001, should only
`be applied to the selections of a specific rare cells in a
`heterogenous population (i.e., PBMCs in blood tumors)
`(Fig. 3D). Without sorting the cells, biopanning was per-
`formed on a glass slide containing the whole cell population.
`This method is advantageous for targeting a lower frequency
`of cells (<0.1% of the total population), as phages that binds
`non-selectively towards the other cells will be screened out.
`Once the phage was bound, UV irradiation was used so that
`the DNA of
`the phage particles on non-target cells is
`crosslinked by UV, while the phage on target cells were
`protected by a minute aluminum disc. Therefore, this method
`ensures that only non-crosslinked phage (target phage)
`were capable of replicating. The disadvantage of this method
`is that it is only optimized for antibody-based ligand selec-
`tion, and thus not suitable for peptide selection. The yield of
`this method averages three antibodies per selection, which
`is very low compared to the other biopanning method (Sor-
`ensen and Kristensen, 2011).
`
`In human screening
`
`To diminish the compatibility of species difference between
`mice and human, phage display had been reported to be
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`Amplified phage display library
`
`A
`
`B
`
`C
`
`D
`
`E
`
`In situ target
`immobilization
`
`In vitro whole cell
`target capture
`
`In vivo target
`capture in tumor
`
`Ex vivo target capture
`in excised tumor
`
`In–human in vivo
`Target capture
`
`Elution of target-specific phages
`
`Infect into bacteria and
`grow colonies on plate
`
`Figure 3. Various approaches in capturing high affinity peptide through phage display screening.
`
`screened against human patients (Fig. 3E). The first in-hu-
`man phage display screening was reported by Arap and
`colleagues in 2002. They reported a heptapeptide SMSIARL
`which could specifically home to prostate vasculature and
`exhibited 10–15 times more specificity to prostate compared
`to other organs (Arap et al., 2002b). Due to their success in
`proving safe usage of phage display in human, FDA
`approved similar techniques to be used by Krag and col-
`leagues to screen tumor-specific peptide via phage display
`screening in terminal stage cancer patients (Krag et al.,
`2006).
`
`TUMOR TARGETING PEPTIDE
`
`Tumor targeting peptide is a powerful tool that could be used
`in cancer diagnosis and treatment (Heppeler et al., 2000) as
`
`they have lower production cost and scale-up, easy to syn-
`thesize and yet they possess most if not all the merits of a
`targeting ligand: high affinity and specificity towards the
`target, with the advantage of high tumor penetration as
`compared to the large-sized antibody-based ligand (AlDe-
`ghaither et al., 2015). In the complexity of solid tumor, a
`peptide could be used to target the malfunctioned tumor
`vasculature, the dense extra-cellular matrix, tumor stromal
`cells, or overexpressed receptor on tumors. Herein, we will
`discuss some prominent examples of peptides identified
`through phage display biopanning techniques and their
`application in the biomedical field.
`
`Peptide targeting tumor-microenvironment (TME)
`
`Tumor microenvironment (TME) is a complex plethora of
`multiple components including tumor-associated vasculature,
`
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`Tumor targeting peptide for cancer therapy
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`REVIEW
`
`A
`
`B
`
`C
`
`Neutrophils
`
`Macrophages
`
`Fibroblasts
`
`T-cells
`
`B-cells
`
`NK cells
`
`Tumor cells
`
`Endothelial cells
`
`Pericytes
`
`Extra-cellular matrix
`
`Figure 4. Major components in the TME. (A) tumor vasculature components and extra-cellular matrix, (B) tumor stromal cells and
`(C) over-expressed receptors on tumor cells.
`
`extra-cellular matrix, cancer associated fibroblast,
`tumor
`associated macrophages,
`immune cells (neutrophils, NK
`cells, T cells, B cells) and tumor cells (Binnewies et al., 2018)
`(Fig. 4). Often, these cells transformed into tumor-like phe-
`notype as tumor progresses. For example, most tumor resi-
`dent macrophages are M2-like (pro-tumoral) which means
`they are programmed to assist in tumor growth rather than
`having an M1-like (anti-tumoral) phenotype (Mantovani et al.,
`2017). These changes could be brought forth by constant
`communication with the other components in the TME through
`autocrine or paracrine manner. Therefore, by identifying
`peptide specific to these TME targets could generate drugs
`homing to TME that could efficiently normalize, modulate or
`disrupt the TME components. There are three points of inter-
`vention, namely (i) targeting tumor vasculature, (ii) targeting
`extra-cellular matrix, (iii) targeting tumor stromal cells (mac-
`rophages, cancer associated fibroblasts etc.).
`
`Peptide targeting tumor vasculature
`
`Angiogenesis is an event of the formation of new blood
`vessels and is vital in the event of tumor growth and pro-
`gression. Due to the continuous formation of new blood
`vessels to feed the tumor, a hyper-vascular tumor could grow
`beyond the size of millimeter in diameter (Bergers et al.,
`1999). Therefore, stopping a tumor’s blood supply can dra-
`matically reduce the tumor growth, and in some cases, even
`resulted in total tumor eradication (Ferrara and Alitalo, 1999;
`O’Reilly et al., 1999). The morphology of tumor vasculature
`is very different from normal tissue vasculature. Due to the
`on-going angiogenesis,
`tumor vasculatures consistently
`express angiogenic marker at high concentration (i.e., inte-
`grins, VEGFR) and are usually tortuous (Bergers et al.,
`
`1999), with pronounced hypoxic region. Tumor vasculatures
`are also “leaky” in nature and this might be related to pericyte
`deficiency (Ruoslahti, 2000),
`therefore Folkman hypothe-
`sized that angiogenesis inhibition could be used to treat solid
`tumors (Folkman, 1971).
`
`Peptide targeting tumor endothelial cells (EC)
`
`The peptides that home to tumor vasculature may also be
`useful in targeting therapies specifically to tumors. Tumors
`are critically dependent on blood supply; therefore, blocking
`or eliminating that supply can profoundly suppress tumor
`growth (Denekamp, 1993; Hanahan and Folkman, 1996;
`Bergers et al., 1999; Jain, 2001). Since blood vessels are
`easily accessible through IV administration, and they do not
`readily acquire mutations as cancer cells that leads to drug
`resistance (Kerbel, 1991; Boehm et al., 1997),
`targeting
`tumor ECs could be a promising approach for targeted drug
`delivery.
`A classic example of vasculature targeting peptide is
`none other than the “RGD” peptide. Rouslahti and col-
`leagues first isolated this peptide by phage display in vivo in
`the form of cyclic peptide CDCRGDCFC (RGD-4C). This
`peptide has been validated to selectively binds αvβ3 and
`αvβ5 integrins (Koivunen et al., 1995); and have shown to
`home to the vasculature of tumors (Pasqualini et al., 1997).
`Interestingly, RGD domain is also vital for the binding of
`vitronectin and fibronectin and to integrins, although it is now
`known that these molecules bind to different subset of inte-
`grin (Ruoslahti, 2003).
`Arap et al. also developed a set of cyclic peptide CNGRC
`sharing “NGR” motifs (Arap et al., 1998). These peptides
`have been shown to bind to tumor vasculatures in breast
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`REVIEW
`
`Phei Er Saw and Er-Wei Song
`
`Peptide affinity (Kd)/
`LC50/IC50
`LC50∼10 mmol/L
`
`LC50∼34–481
`mmol/L
`
`ND
`
`ND
`
`ND
`
`ND
`IC50∼10 mmol/L
`IC50∼10 mmol/L
`ND
`
`ND
`Kd∼11 µmol/L
`Kd∼65 nmol/L
`4.58 ± 1.4 µmol/L
`
`ND
`
`MC = 0.70
`
`MC = 0.74
`
`ND
`
`IC50∼0.01–10
`mmol/L
`
`Reference
`
`(Koivunen et al.,
`1995)
`
`(Pasqualini et al.,
`2000)
`
`(Essler and
`Ruoslahti, 2002)
`
`(Arap et al., 2002a)
`
`(Ndinguri et al.,
`2012)
`
`(Burg et al., 1999)
`
`(Han et al., 2015)
`
`(Kim et al., 2012b)
`
`(Kim et al., 2012a)
`
`(Cieslewicz et al.,
`2013)
`
`(Brinton et al., 2016)
`
`(Landon and
`Deutscher, 2003)
`
`(Goodson et al.,
`1994)
`
`Table 2. Peptide targeting TME and TME stromal cells
`
`Target
`
`Peptide sequence
`
`Endothelium/av integrin
`
`ACDCRGDCFCG (‘‘RGD’’ motif)
`
`Endothelium expressing
`aminopeptidase N/CD13
`
`Breast endothelium/amino
`peptidase P
`
`Prostate endothelium
`
`Lung endothelium (membrane
`dipeptidase)
`
`CNGRC
`
`CPGPEGAGC
`
`SMSIARL
`
`CGFECVRQCPERC
`
`Skin endothelium
`
`CVALCREACGEGC
`
`CRRHWGFEFC
`
`CTTHWGFTLC
`
`TAASGVRSMH
`
`LTLRWVGLMS
`
`CTVRTSADC
`
`HCSSAVGSWTWENGKWTWKGIIRLEQ
`
`FHKHKSPALSPV
`
`YEQDPWGVKWWY
`
`HTTIPKV
`
`APPIMSV
`
`MMP9
`
`MMP2
`
`Transmembrane chondroitin sulfate
`proteoglycan NG2
`
`Tumor-associated FN
`
`Tenascin C
`
`Tumor associated macrophages
`(TAMs)
`Cancer associated fibroblasts (CAFs)
`
`Protein&Cell
`
`Urokinase plasminogen activator (uPA)
`receptor (uPAR)
`
`AEPMPHSLNFSQYLWYT
`
`LWXXAr (Ar = Y, W, F, H) XFXXYLW
`
`carcinoma, melanoma and Kaposi’s sarcoma (Pasqualini
`et al., 1997; Arap et al., 1998; Pasqualini et al., 2000).
`Subsequently, many other publications followed, describing
`the isolation of tumor vasculature related targeting peptides
`(Table 2) (Landon and Deutscher, 2003; Zurita et al., 2003;
`Ruoslahti, 2004; Kelly et al., 2005; Su et al., 2005).
`
`Peptide targeting MMPs
`
`family is among the
`Matrix metalloproteinases (MMPs)
`molecules that are upregulated in tumor microenvironment,
`and has been known to be functionally important in angio-
`genesis (Koivunen et al., 1999). Not only that, MMPs are
`also involved in increasing cell motility and invasiveness
`(Birkedal-Hansen, 1995). Although MMPs are secreted
`proteins, they are able to mediate phage homing. This might
`be due to the binding of MMP-2 and MMP-9 to αvβ3 integrin
`(Brooks et al., 1996),
`thus forming a complex that
`is
`stable enough for the binding of phage. Apparently,
`the
`complex is stable enough for strong binding of the phage to
`the MMP. Interestingly, the selected phage bound to MMP-2
`and MMP-9 also specifically homes to tumor vasculature
`
`(Koivunen et al., 1999), indicating that (i) that one, or both, of
`these MMPs is specifically expressed in tumor vasculature
`and (ii) they are available for phage binding from the circu-
`lation. Multiple peptides inhibiting MMP families have been
`isolated through phage display screening. Their sequence,
`activities and function are summarized in Table 2 (Ujula
`et al., 2010; Ndinguri et al., 2012).
`
`Peptide targeting pericytes of angiogenic vessels
`
`Pericytes secrete growth factors that stimulate EC prolifer-
`ation. Pericytes also secrete proteases to modulate the
`surrounding ECM and guide EC migration (Gerhardt and
`Betsholtz, 2003; Armulik et al., 2005; Saunders et al., 2006;
`Stapor et al., 2014). Recently, more researches are pointing
`towards the importance of pericyte coverage in vessel
`remodeling, maturation, and stabilization (Ribeiro and Oka-
`moto, 2015). Therefore, pericyte might be the overlooked
`player in angiogenesis and should be given more emphasis
`in anti-tumor targeted therapy.
`Several rounds of biopanning led Burg et al. to identify
`two decapeptides (TAASGVRSMH and LTLRWVGLMS)
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`Tumor targeting peptide for cancer therapy
`
`REVIEW
`
`specific to a transmembrane chondroitin sulfate proteogly-
`can NG2, which is expressed in pericytes of angiogenic
`vessels (Schlingemann et al., 1990; Burg et al., 1999).
`These peptides specifically homed to tumor vasculature
`in vivo but not to tumor vasculature in NG2 knockout mice,
`indicating the specificity and targeting capability of these
`peptides (Burg et al., 1999). Although the role of NG2 in
`angiogenesis is still unclear, NG2 is a cell surface receptor
`for type-VI collagen and also binds to PDGF-A, which could
`potentially stimulate this growth factor (Nishiyama et al.,
`1996). As a component in pericyte, NG2 is undetectable in
`endothelial cells (Burg et al., 1999), therefore blocking NG2
`represents a specific pericyte targeting.
`
`Peptides targeting extra-cellular matrix (ECM)
`
`The role of ECM components is now recognized as an
`important determinant in the growth and progression of solid
`tumors (Wernert, 1997; Pupa et al., 2002). ECM is exten-
`sively remodeled in tumor progression through 2 main pro-
`cesses:
`(i) neosynthesis of ECM components
`(i.e.,
`alternative splicing mechanism of fibronectin to include EDA
`and EDB domain in malignant tumor fibronectin) and (ii)
`degradation of ECM by hydrolytic enzymes (e.g., proteases)
`that are produced, activated or induced by neoplastic cells,
`therefore become more permissive environment for tumor
`growth (Kaspar et al., 2006).
`Tumor-associated fibronectin Fibronectin serves as a
`coordinator between cancer cells and ECM, and is involved
`in cancer cell survival, proliferation, invasion and metastasis
`(Wierzbicka-Patynowski and Schwarzbauer, 2003). One of
`the most extensive changes in ECM remodeling is the
`addition of extra-domain A and B (EDA and EDB), which are
`alternatively spliced-in during the synthesis of tumor-asso-
`ciated fibronectin. These domains are undetectable in
`healthy adult but has been found in high concentrations in
`malignant tumors. Clinical evidences indicated that tumor-
`associated FN (also termed oncofetal FN), is overexpressed
`in many malignant cancers, including breast cancer (Ioachim
`et al., 2002; Bae et al., 2013), prostate cancer (Suer et al.,
`1996; Albrecht et al., 1999), bladder cancer (Arnold et al.,
`2016), oral squamous cell carcinoma (Lyons et al., 2001),
`head and neck squamous cell carcinoma (Mhawech et al.,
`2005), colorectal cancer (Inufusa et al., 1995) and lung
`cancer (Khan et al., 2005), and upregulated FN expression
`has been correlated with poor prognosis of the patients.
`Therefore, tumor-associated FN represents an ideal target
`for solid tumor targeting.
`Through in situ phage display technology, Kim et al.
`developed an EDB binding scaffold-like peptide termed
`APTEDB (Kim et al., 2012b). APTEDB consists of a stabilizing
`scaffold and two target-binding regions, mimicking the mor-
`phology of a DNA leucine zipper. Taking advantage of the
`synergistic three-dimensional structure for optimal binding,
`APTEDB exhibited a high binding affinity (Kd ∼65 nmol/L) to
`EDB and could be used as a targeting ligand to be
`
`conjugated to anti-cancer drugs for high tumor selectivity
`and reducing systemic toxicity (Kim et al., 2014; Kim et al.,
`2016), deliver biologics (i.e., oligonucleotides, siRNA and
`drugs) for solid tumor treatment (Saw et al., 2013; Saw et al.,
`2015; Saw et al., 2017) and to encapsulate superparamag-
`netic iron oxide particles for Magnetic Resonance Imaging of
`EDB over-expressing tumors (Park et al., 2012). In another
`study, Han et al. developed a cyclic nonapeptide (ZD2) with
`the sequence of CTVRTSADC that could be used for EDB
`specific targeting and imaging of prostate cancer. This linear
`peptide, which has Kd ∼11 μmol/L binding affinity towards
`EDB, demonstrated excellent specific targeting to prostate
`cancer in vivo and could be utilized as an imaging agent for
`EDB-overexpressing prostate cancer (Han et al., 2015).
`Tenascin C (TNC) TNC is a glycoprotein which forms a
`large structure body by assembling other ECM molecules
`and participates in cell adhesion, movement, permeation,
`survival, migration and differentiation (Chiquet-Ehrismann,
`1990). As with tumor-associated FN, TNC is not usually
`expressed in normal cells except in immune tissues, such as
`bone marrow and thymus gland (Klein et al., 1993; Heme-
`sath and Stefansson, 1994), but is specifically expressed in
`malignancy, inflammation and wound healing. It had been
`reported that the elevated expression of TNC depended on a
`malignancy in the tumor stroma of some malignancies,
`including oral cancer, sarcoma, breast cancer, and colon
`cancer, squamous cell carcinoma (Hindermann et al., 1999)
`chondrosarcoma (Ghert et al., 2001), breast cancer (Tsun-
`oda et al., 2003) and colon cancers (Hanamura et al., 1997;
`Suzuki et al., 2017).
`Kim et al.
`isolated a peptide that not only selectively
`bound to TNC in xenograft mouse tissue and patient tumors
`but also reduced TNC-induced cell rounding and migration.
`Due to the bulky size of TNC, they adopted two independent
`the first using full-length TNC (expressed in
`screening;
`eukaryotic cells) and the second using alternative spliced
`domain (expressed in bacteria). Out of a total of 35 clones,
`19 had the same sequences (denoted peptide #1,
`FHKHKSPALSPV, 54.2% consensus) and another 13 clones
`were also identical (denoted peptide #2, FHKPFFPKGSAR,
`37.1% consensus). The binding affinity of peptide #1 to TNC
`was 4.58 ± 1.4 µmol/L (Kim et al., 2012a).
`
`Peptide targeting tumor associated macrophages
`(TAMs)
`
`High density of TAMs has been correlated to poor prognosis
`in several types of cancers, including brain, breast, ovarian
`and pancreatic cancers, where the majority of these TAMs
`express M2-like phenotype (Kurahara et al., 2011; Medrek
`et al., 2012; Colvin, 2014; Zhou et al., 2015). Therefore, M2-
`like TAMs have been exploited as therapeutic targets, and
`positive outcomes were shown in selective depletion of this
`macrophage subpopulations (Georgoudaki et al., 2016).
`Small molecules such as folic acid (targeting folate receptor
`
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`REVIEW
`
`Phei Er Saw and Er-Wei Song
`
`β) and mannose (targeting mannose receptor) have been
`conjugated to drugs or carriers for macrophage targeting and
`drug delivery (Hashida et al., 2001; Low et al., 2008; Yu
`et al., 2013). However, these receptors are not macrophage
`specific and they are also expressed in other cell types for
`example, mannose receptors are also expressed in dendritic
`cells (Sallusto et al., 1995)). Folic acid also binds different
`isoforms of
`folate receptors on tumor cells and normal
`epithelial cells (Ross et al., 1994), therefore diminishing the
`specificity effect of the ligand. In 2012, Segers et al. reported
`a novel peptide that binds selectively to scavenger receptor-
`A on macrophages in atherosclerotic plaques. Nevertheless,
`it was found that this receptor is also expressed on dendritic
`cells (Segers et al., 2012). Therefore, M2-like macrophage-
`specific peptide should be screened and developed for
`clinical application.
`Cieslewicz et al. polarized murine bone marrow-derived
`macrophages with either IFN-γ and LPS or with IL-4 to
`generate both M1 and M2 cells for biopanning. After three
`rounds of phage panning, highly selective M2 macrophage-
`binding peptides were identified, and this peptide binds
`preferentially to M2 cells. Sequencing of
`the 10 clones
`obtained
`above
`revealed
`two
`unique
`sequences:
`YEQDPWGVKWWY (denoted M2pep Phage, consensus
`80%), and HLSWLPDVVYAW (consensus 20%). M2pep
`Phage demonstrated higher affinity and selectivity towards
`M2; 10.8-fold higher binding to M2 macrophages over
`scramble-M2pep, as well as 5.7-fold higher binding to M2
`over M1 macrophages. Furthermore, after
`intravenous
`administration, M2pep Phage was able to selectively binds
`M2-like TAMs in mouse colon carcinoma tumors (Cieslewicz
`et al., 2013).
`
`Peptide targeting cancer associated fibroblasts (CAFs)
`
`One of the dominant cell type in solid tumor is CAFs (Aug-
`sten, 2014). They are likely to be derived from the mesoderm
`and exhibited mesenchymal-like features (Kalluri and
`Weinberg, 2009). CAFs are often found in close vicinity or in
`direct contact with tumor cells (Kalluri and Weinberg, 2009).
`In normal condition, fibroblasts are likely to be quiescent or in
`resting state, yet became activated in response to growth
`factors, cytokines and mechanical stress (Kalluri and Wein-
`berg, 2009; Rasanen and Vaheri, 2010; Shiga et al., 2015).
`Unlike tumor cells that presents diverse marker proteins on
`cell surface, CAFs selectively overexpressed certain pro-
`teins, such as fibroblast-activated protein-α (FAP-α) and α-
`smooth muscle actin (α-SMA) (Bhowmick et al., 2004; Kalluri
`and Zeisberg, 2006; Franco et al., 2010; Rasanen and
`Vaheri, 2010). Therefore, CAF targeting or
`responsive
`nanomaterial may be an efficient strategy to achieve
`improved antitumor efficacy.
`Brinton et al. presented a new strategy for analysis by
`