Peptides derived from extracellular matrix proteins
The extracellular matrix (ECM) is a network of fibrous proteins that surrounds cells and provides a conduit for cellular communication. Also, the ECM serves as a signaling hub for cells, via direct interaction with cells or through different soluble factors trapped in the network. Hence, protein components of this network have been investigated for potential smaller peptides that can elicit or interfere with specific cellular responses, such as cellular migration, proliferation or survival.
A well-studied integrin-binding motif, the tri-amino acid peptide RGD, is derived from fibronectin and is capable of controlling cellular behavior, particularly adhesion and migration. Integrins are heterodimeric membrane receptors composed of non-covalently associated subunits α and β, and demonstrating cationic-dependent interaction with multiple ECM proteins. The integrin family is an extensive group of cellular receptors, generated by the association of one of the 18α subunits with one of the 8β subunits generating 24 unique heterodimers, involved in attachment and migration of cells to the surrounding ECM. Some integrins such as α5
can recognize single ligands while others such as αV
can bind multiple ligands. Binding of a ligand to the extracellular domain leads to integrin clustering and subsequent intracellular signal transduction activation. Unlike growth factors, integrin signaling is not based on intrinsic enzymatic activation; rather, it is dependent on co-clustering with kinases and adaptor proteins within focal adhesion complexes. In vitro
and in vivo
experiments have identified a number of endothelial cell integrins involved in cell growth, survival and migration during angiogenesis. These integrins include fibronectin-binding (α5
) and laminin-binding (α1
was the first integrin shown to regulate angiogenesis; it is present on tumor blood vessels but not on normal tissues and antagonists have been shown to inhibit angiogenesis and tumor growth in a variety of animal models of cancer, probably by inducing endothelial cells apoptosis and balancing opposing signals in the tumor microenvironment [22
Cyclization and modifications of the RGD peptide fragment led to the development of an optimized sequence (c-[Arg-Gly-Asp-DPhe-(NMeVal)]), cilengitide, which demonstrates increased potency and specificity towards endothelial cells making it a therapeutic drug candidate. Data on cilengitide and other peptides derived from ECM are presented in , where in vivo
doses are included. In pre-clinical studies it reduces EC proliferation and migration [23
]. The targets for cilengitide are the integrins αv
and since these integrins are also present on some tumor cells, this anti-angiogenic treatment also showed a direct anti-tumorigenic effect specifically in glioma cells[23
]. Besides identifying the target, understanding the mode of action (MOA) of the peptide becomes a very important component in developing successful treatment plans based on the activation or inhibition of particular pathways [23
]. Cilengitide has also been tested in vivo
in several phase II trials of different cancers including prostate cancer [25
], chemotherapy refractory renal, colorectal, melanoma cancers [26
], and glioblastoma [27
]. The treatment was well tolerated and showed a 6-month progression-free survival (PFS) of 15% of the patients and median overall survival (OS) of 9.9 months in the glioblastoma study. Although microvascular density (MVD) was intended as a biomarker it was eliminated due to high tumor variability. In other cancer models, such as prostate cancer, the treatment was well tolerated but the treatment arms did not demonstrate a significant improvement [25
]. This partial failure could be due to the redundancy in signaling pathways present in tumors and high tumor-to-tumor variability influenced by different tumor microenvironments.
Peptides derived from extracellular matrix proteins
RGD modifications have also been undertaken to create targeting molecules. For example, coupling the RGD molecule to a heparin derivative which has been shown to target dividing tumor endothelial cells resulted in an increase in potency in comparison to the unmodified cyclic-RGD. This new construct showed potent inhibition of proliferation, migration, tube formation of EC and of tumor growth in a mouse colon cancer model, accompanied by a reduction in MVD [28
ATN-161, a peptide derived from the synergy region of fibronectin that binds αv
, has been demonstrated to inhibit growth and metastasis in a breast cancer model accompanied by a decrease in MVD (43%) along with inhibition of tumor cell growth via an integrin mediated signaling [29
]. Furthermore, ATN-161 was evaluated in vivo
in several therapy-refractive cancer models. The treatment was well tolerated, and 23% of the patients demonstrated stable disease for >4 months [30
]. The effect of ATN-161 was also investigated in combination with standard chemotherapy treatment in a murine model of liver metastasis. The treatment showed a decrease in tumor burden; a decrease in the number of metastatic lesions and in microvascular density [31
]. A labeled analog of ATN-161, ATN-453, was shown to bind selectively to the neovasculature and not to the pre-existing vasculature in a syngeneic mouse model of adenocarcinoma [32
]. Several clinical trials are investigating the potency of this agent as a monotherapy or in a combination therapy in advanced renal cell carcinoma and recurrent glioma.
In contrast with the RGD-inspired peptides, which were developed by starting with a known peptide sequence by adding structural modifications, such as cyclization and non-natural amino acids, there are peptides that were derived by truncations of longer ECM proteins. Tumstatin, a 28 kDa fragment from the noncollagenous domain of type IV collagen, has been demonstrated to exhibit anti-angiogenic properties [33
]. Further study of this fragment led to the identification of shorter anti-angiogenic fragments [34
]. Structure activity studies resulted in the identification of amino acids responsible for the anti-angiogenic characteristics of the short tumstatin peptide. This novel 22 amino acid peptide demonstrated potency in inhibiting proliferation and migration of EC along with strong inhibition of tumor growth in animal models (Lewis lung carcinoma, SCC-PSA1 teratocarcinoma, and human renal clear cell carcinoma) [36
Thevenard et al.
demonstrated that a shorter fragment of the tumstatin sequence, YSNS, forms a β-sheet which is important for biological activity and that a cyclized version of YSNSG formed a very stable and active structure [37
]. This compound showed activity in inhibiting adhesion and migration of endothelial and melanoma cells. Also this cyclized peptide reduced tumor size, growth and tumor microvasculature in a melanoma model.
Karagiannis and Popel created a bioinformatics-based methodology to identify novel putative anti-angiogenic fragments based on homology to known anti-angiogenic sequences. This effort was based on the identification of common motifs present in known anti-angiogenic peptides which were then used to identify other endogenous proteins that contained the motif. Thus, multiple peptide fragments were identified, in particular 10 peptides belonging to the class of collagen IV. One candidate which was derived from the α5 fibrils of type IV collagen and is termed pentastatin-1 showed significant activity inhibiting proliferation and migration of EC cells [39
]. This peptide has been tested in mouse xenograft models of breast cancer [40
] and small cell lung cancer [41
] and showed significant inhibition of tumor growth and MVD.
Endostatin, which was first isolated in 1997, is a 20 kDa protein fragment of the extracellular matrix protein collagen XVIII that successfully inhibited Lewis lung cancer metastases [42
]. As an early-identified endogenous inhibitor of angiogenesis with strong potential, endostatin was rapidly moved to clinical trials; however poor solubility and difficulty purifying the molecule through recombinant expression made it inappropriate for the clinic [43
]. Endostar, a modified form of the molecule, containing a MGGSHHHHH tag added to the N-terminal, was approved in 2005 in China for non-small cell lung cancer (NSCLC) [44
]. Endostar has been tested in tumor xenografts of human nasopharyngeal carcinoma and human lung adenocarcinoma each in combination with radiation, where it significantly increased endothelial cell apoptosis and limited tumor growth [45
]. Endostar is a large polypeptide, and must be produced recombinantly in E-coli for quantities sufficient for clinical trials.
Shorter fragments of endostatin have also been identified and tested in pre-clinical cancer models. A 27-amino acid zinc binding fragment of endostatin from the N-terminal called mP1 was isolated and shown to retain the anti-tumor, anti-migratory, and anti-permeability activities of endostatin. This peptide fragment was tested in BxPC-3 and Lewis lung carcinoma models where it demonstrated significant tumor suppression [46
]. Morbidelli et al.
segmented endostatin into five fragments named I(1–39), II(40–89), III(90–134), IV(135–184) and IVox(135–184), which contains a disulfide bond between C135–C165. Fragment IV and IVox exhibited anti-angiogenic activity similar to native endostatin, and inhibited tumor growth in an A4-431 xenograft model [47
]. Similarly, Cattaneo et al.
created fragments of endostatin corresponding to residues I(6–49), II(50–92), III(93–133), IV(134–178). They found fragment I to be a potent inhibitor of angiogenesis in vitro
and in vivo
in Matrigel plug assays [48
]. Olsson et al.
demonstrated the minimal active epitope of endostatin to be a heparin-binding motif of 20 amino acids (180–199, FLSSRLQDLYSIVRRADRAA), which was effective in human tumor xenografts [49
]. Using crystal structure analysis, Wickstrom et al.
created five endostatin-derived sequences of 11–13 amino acids, and found a minimal epitope (IVRRADRAAVP) that inhibited endothelial cell migration and tube formation. However, its activity in vivo
is unknown [50
Other ECM-derived peptides include laminin-derived peptides, many of which are pro-angiogenic. Two peptides of interest are A13 (RQVFQVAYIIIKA) and C16 (KAFDITYVRLKF), derived from the alpha 1 and gamma 1 domains of laminin. For unknown reasons a scrambled sequence of C16, termed C16S peptide (DFKLFAVTIKYR) inhibited angiogenesis and a further mutation from T to Y termed C16Y (DFKLFAVYIKYR) had stronger effects than C16S in cell adhesion assays and inhibited tumor growth in breast xenograft model [51
Peptides derived from growth factors and growth factor receptors
Vascular endothelial growth factor (VEGF) is one of the most important modulators of angiogenesis. Inhibiting its interaction with the receptor via antagonistic peptides could present an effective anti-angiogenic therapy. Studying the binding interaction of VEGF8–109
with VEGF receptor-1 (VEGFR1), one of the VEGF-A binding partners, resulted in the identification of critical residues responsible for binding. Coupling this information with Alanine-scan analysis aided the development of several potent drug candidates. Data on peptides derived from VEGF and other growth factors are presented in . Recently, the short sequence CPQPRPLC was identified through a subtractive-phage display screening approach as a sequence targeting VEGFR1 and neuropilin-1. The shorter epitope RPL was identified as the minimal sequence required for activity [53
]. This sequence was retro-inverted D
(LPR), and displayed activity in a syngeneic mouse mammary cancer model [54
]. This may be the only known compound that targets both VEGFR1 and neuropilin-1.
Peptides derived from Growth Factors or their receptors
A peptide derived from the 6a-exon domain of the VEGF gene demonstrated activity in binding to HUVEC surface by competing binding of the VEGF165
to cell surface. The 20 amino acid peptide inhibited cell migration and tumor growth in lung cancer model by reducing microvascular density [55
Fibroblast growth factor (FGF), another modulator of angiogenesis, is involved in angiogenesis and anti-angiogenic therapies. Contrary to initial expectations that anti-angiogenic therapies would not be susceptible to drug resistance, resistance to anti-VEGF treatment is observed, notably due to upregulation of FGF2. Therefore, antagonists, such as peptides derived from long-pentraxin-3 (PTX3) which bind FGF2 and prevent it from binding to its receptor are being pursued as anti-angiogenic agents. Several peptides derived from PTX3 have been investigated and found to inhibit the proliferation and adhesion of EC. They also inhibit angiogenesis in the chick embryo chorioallantoic membrane assay (CAM) assay and tumor xenograft growth in zebrafish [56
Transforming growth factor β (TGFβ) is a pleiotropic agent and its effects are context driven. In early phases of tumor development TGFβ exhibits anti-angiogenic activity, in later stages it appears to mainly have pro-angiogenic activity. Several peptides have been studied, however a 14 amino acid peptide (P144) derived from TGFβ type III receptor displaying a high affinity for soluble TGFβ has been shown to reduce skin fibrosis and colonization of the bone by lung cancer cells [58
]. Serratti et al.
has investigated its anti-angiogenic potential and demonstrated it to be capable of inhibiting tube formation of EC in vitro
and in vivo
in the Matrigel sponge assay. P144 appears to exert its anti-angiogenic effect by inhibiting the signaling and downregulation of the pro-angiogenic response initiated by TGFβ [59
Peptides derived from coagulation cascade proteins
The coagulation cascade along with angiogenesis and homeostasis are among the host responses associated with cancer [60
]. Several proteins involved in the coagulation cascade have been shown to exhibit potent anti-angiogenic and anti-neoplastic activity; anti-angiogenic peptides derived from these proteins are presented in .
Peptides derived from proteins involved in the coagulation cascade
High molecular weight kininogen (HK) is a multifunctional protein that plays a role in multiple pathophysiological conditions such as thrombosis and inflammation. It also binds to EC where it can be cleaved by plasma kallikrein. The cleaved HK has been shown to inhibit migration and proliferation of EC [61
]. Proteolytic cleavage of HK releases a vasoactive 9 amino acid (nonamer) peptide called bradykinin that appears to be involved in the coagulation cascade and have pro-angiogenic activity. B9870, a peptide antagonist of bradykinin has been investigated as a therapeutic agent in combination with chemotherapy in lung and prostate cancer. Treated tumors showed a reduction in microvasculature and increased number of apoptotic cells indicating this compound has application in cancer therapy by inhibition of angiogenesis [62
Histidine-proline-rich glycoprotein (HPRG) is a potent anti-angiogenic protein evolutionarily related to high molecular-weight kininogen [63
]. HRGP330 is a 35 amino acid sequence first identified by Dixelius et al.
from the histidine/proline-rich domain, and is a potent inhibitor of angiogenesis for pancreatic carcinoma [64
]. A synthetic peptide (HHPHG)4
binds to tropomyosin and inhibits angiogenesis in vitro
and in vivo
in syngeneic mouse tumor models [65
]. The peptide functions by targeting focal adhesions in endothelial cells and disrupting cytoskeletal organization [66
], is a biologically vasoactive peptide. Substitution of D-Ala at position seven created [D-Alanine7]-Ang-(1–7), A-779, a compound that inhibits EC tube formation and results in microvascular density reduction in treated lung xenografts [67
]. In Phase I clinical trials, subcutaneous administration of 400μg/kg daily for 5 days in 3 cycles of treatment lead to stable disease for >3 months in most patients. Measuring the efficacy of treatment via a biomarker is very useful in clinical settings and in most cases has not been analyzed. The clinical trial discovered that plasma levels of placental growth factor (PlGF) can serve as a biomarker for treatment responsiveness [68
Kringles are large protein domains stabilized by disulfide bonds important in the blood coagulation cascade. KV11 a 12-mer (dodecamer) derived from Kringle domain 5 has been shown to inhibit angiogenesis in vitro
by inhibiting EC migration and microtubule formation. The peptide has been shown to inhibit capillary network formation in vivo
and limit tumor growth in breast cancer xenografts by decreasing microvascular density through the c-SRC/ERK pathway [69
Urokinase plasminogen activator system (uPA system) is comprised of 4 members, urokinase plasminogen activator, its receptor, and two plasminogen activator inhibitors. It has been demonstrated that the uPA system is involved in cancer progression, in particular in the remodeling of ECM by modulating cell adhesion. Clinical studies showed that either inhibition of uPA activity or its binding to the receptor reduced tumor growth, angiogenesis and metastasis [70
6, an octamer capped peptide derived from the urokinase plasminogen activator, has been demonstrated to inhibit migration of EC while having no effect on proliferation. However, in vivo
in combination with cisplatin,
6 demonstrated significant tumor growth reduction in a subcutaneous glioma model [71
], and in combination with tamoxifen demonstrated strong activity in orthotopic breast cancer xenografts [72
Fibrinogen, a glycoprotein composed of the pairs of non-identical chains, gets cleaved by thrombin to form fibrin, the main component of blood clots, upon vascular injury. Various cleavage products of fibrinogen are involved in regulating cell adhesion and spreading. A 20 amino acid peptide derived from the β chain of the E-fragment of fibrinogen has been reported to inhibit the binding of EC to collagen IV via the αV
integrin. This peptide does not inhibit proliferation or migration of EC. However, it significantly inhibits the adhesion of EC cells to collagen IV and disrupts vessels formation; in a breast cancer xenograft model the peptide showed activity in reducing tumor growth [73
Peptides derived from chemokines
Platelet growth factor 4 (PF4) is a cytokine and part of the CXC chemokine family. When the ELR motif precedes the CXC pattern the chemokine is usually pro-angiogenic and if the motif is absent it is usually anti-angiogenic, the exception being the growth related protein β which contains the ELR motif but has been shown to possess anti-angiogenic properties in vitro
and in vivo
]. We present peptides derived from several chemokines in . Replacement of the DLQ amine terminal motif with the ELR motif and its consequent DLR mutation in the PF447–70
fragment generated peptides with anti-angiogenic properties that abrogated VEGF or FGF2 induced proliferation, and inhibited capillary network formation in CAM assays. Also, intratumoral delivery of these peptides via Alzet pumps in an intracranial glioma model strongly inhibited tumor growth [75
]. Another variant of the same PF4 fragment was shown to inhibit melanoma xenografts at low doses (7 μg overall treatment) [76
]. Sustained delivery via nanospheres delivered intratumorally in orthotopic glioma models showed nanoparticles homed around the tumor with considerable amount of peptide detected even after 14 days after a single intratumoral injection [77
Peptides derived from chemokines
Using a bioinformatics methodology, a group of 7 anti-angiogenic peptides derived from ELR positive CXC chemokines has been identified. They inhibit migration and proliferation of EC [78
]. One peptide from the group, CXCL1 derived chemokinostatin-1, was also tested in breast cancer xenografts where it exhibited tumor growth inhibition accompanied by a decrease in microvascular density [40
An analysis of the 3D structure of PF4, an anti-angiogenic protein, revealed the importance of a β-sheet structure which appeared to be critical for activity. Based on this information, a 33 amino acid peptide, anginex, was developed to capture the critical conformation. This peptide showed significant inhibition of EC proliferation, migration, and capillary network formation in the CAM assay [79
]. Treatment of ovarian tumor xenografts with anginex (10mg/kg/day) via a pump significantly inhibited tumor growth. The peptide was efficacious in both preventing tumors from forming and inhibiting the growth of pre-existing tumors and it also resulted in a decrease in intratumoral microvasculature [80
]. Anginex-conjugated liposomes have also been shown to be capable of targeting fluorescently labeled paramagnetic liposomes to activated endothelium [83
]. Moreover, treatment with anginex proved to be efficient in delaying tumor growth post radiation therapy [84
Peptides derived from Type I Thrombospondin domain containing proteins
Thrombospondin (TSP) was the first identified endogenous inhibitor of angiogenesis [86
]; peptides derived from its family are presented in . The TSP family is composed of five different polypeptides named thrombospondin 1–5 with TSP1 and TSP2 being similar in structure [87
]. The anti-angiogenic activity of TSP1 is localized to three type I repeats in the pro-collagen domain, and several peptides derived from these regions have been identified as angiogenesis inhibitors. The use of full-length TSP1 has been prohibitive due to its size and other multiple biological functions. Substitution of D-enantiomers of any of 3 L-amino acids of the non-active 19-mer peptide from the Mal II sequence conferred activity approaching that of the full length TSP-1 protein. The peptide containing D-Ile at position 15 could be shortened to a seven amino acid sequence without loss of activity [88
]. A more solubilized form of this molecule, DI-TSPa, demonstrated tumor growth inhibition in a human bladder cancer model, and inhibition of melanoma metastases in vivo
Peptides derived from TSP1 domain containing proteins
Further developed thrombospondin-derived molecules include ABT-526 and ABT-510. Based on a heptapeptide of the known anti-angiogenic thrombospondin-1 repeat, various amino acid substitutions were made to improve the PK/PD profile. ABT-526 and ABT-510 were selected from hundreds of variations, and both inhibit angiogenesis in vitro
and in vivo
]. Further toxicity data in dogs with naturally occurring cancers showed both ABT-526 and ABT-510 were well tolerated [91
]. Clinically, ABT-510 has been tested in renal cell carcinoma [92
], soft tissue sarcoma [93
], and glioblastoma [94
]. The drug was well tolerated but in phase II trials for renal carcinoma and soft tissue sarcoma the overall efficacy of the monotherapy was not significant.
Properdin is a plasma protein active in the alternative complement pathway of the innate immune system and contains a conserved TSP1 domain [95
]. Properdistatin, a bioinformatically-identified peptide derived from the TSP1 domain of properdin, has been demonstrated to exhibit potent anti-angiogenic properties in vitro
], and in breast cancer xenografts [40
Peptides derived from serpins
Serpins are a group of proteins with similar structure able to inhibit proteases. Proteins from this family exhibit anti-tumor activity [97
]; peptides derived from this family have been tested in cancer as anti-angiogenic agents as shown in .
Peptides derived from serpin proteins
Pigment epithelium-derived factor (PEDF) is a potent anti-angiogenic, non-inhibitory serpin protein first identified in 1991 as having potent anti-angiogenic attributes greater than those of endostatin [98
]. Although a significant extent of PEDF research has focused on ocular applications, PEDF loss has been correlated with the growth of several types of tumors including prostate cancer, pancreatic cancer, osteosarcomas, breast, neuroblastomas, melanomas and gliomas [99
]. Overexpression of the native PEDF molecule delays the growth and progression of solid tumors through p53 mediated apoptosis [100
]. Its anti-angiogenic properties have been localized to an N-terminal fragment TGA (16–26) and an overlapping 34 amino acid peptide (24–57) that suppresses PC-3 cell prostate tumor growth and microvascular density. A separate 44 amino acid epitope (58–101) is responsible for neurotrophic activity, but it contains a region called the ERT segment (78–94) which is anti-angiogenic [101
The 34-mer anti-angiogenic peptide contained in native PEDF was further truncated to identify the minimal active epitope for anti-angiogenic activity. A P18 fragment blocked angiogenesis in vivo
in renal cell carcinoma and prostate cancer xenografts, even more effectively than the 34 amino acid epitope and native PEDF [102
]. Ek et al.
created four synthetic 25-mer PEDF peptides termed StVOrth-1, -2, -3, and -4 with StVOrth-2 (residues 78–102) and StVOrth-3 (residues 90–114) inhibiting osteosarcoma growth and pulmonary metastases [103
]. Other serpin-derived proteins such as maspin are also potent inhibitors of angiogenesis; however, to our knowledge there are no peptides from this family tested in cancer models.
Peptides derived from other proteins
Troponin I is an intracellular protein responsible for binding actin in myofilaments. A 19 amino acid sequence derived from bovine and shark cartilage has demonstrated anti-angiogenic properties including limiting endothelial cell tube formation, and preventing liver metastases after injection of CAPAN-1 pancreatic cancer cells in mice [104
]; data about this peptide along with other peptides that possess anti-angiogenic potential in cancer models are presented in . The HPRG derived peptide and the 19-mer derived from troponin I are unique as they both have intracellular targets while the other peptides discussed in this review are thought to work by binding to cell membrane receptors or extracellular pro-angiogenic factors.
Peptides of various origin with anti-angiogenic activity in tumors
Bombesin, a tetradecamer which stimulates gastrin release has also been shown to possess anti-neoplastic properties. Bajo et al.
also demonstrated that a shorter fragment, RC-3940 II, a modified 11-mer has anti-angiogenic properties. Daily administration of 10μg significantly inhibited tumor growth accompanied by a decrease in tumor mRNA levels of FGF2, IFGF2, VEGF-A and a reduction in tumor vasculature [105
PAMP, a 20 amino acid peptide derived from the amine terminus of Proadrenomedullin exhibits very strong angiogenic activity (at concentration six orders of magnitudes lower than VEGF). Interestingly, a fragment of PAMP, the amino acids 12–20 generates a peptide that exhibits anti-angiogenic activity. Martinez et al.
demonstrated activity in inhibition of capillary network formation in CAM assay and microvasculature formation in DIVAA. Furthermore, the peptide demonstrated strong activity in inhibiting tumor growth [107
IM-862 is a di-amino-acid peptide (EW) that has anti-angiogenic and immunostimulatory activity in renal cancer carcinoma. Phase II studies of intranasally delivered peptide showed no significant toxicity. The anti-angiogenic effects were measured by monitoring the plasma levels of VEGF and showed a significant decrease as the result of the treatment. However, the efficacy was modest with 7 patients showed no disease progression while 17 progressed on treatment.
Aβ, a dodecameric peptide derived from full length β-amyloid peptide was demonstrated to be effective in tumor suppression in breast xenografts of MCF-7 cells. The peptide showed no cytotoxic or antiproliferative effects on MCF-7 cells in vitro, however the reduction in proliferation in vivo is attributed to the anti-angiogenic effects of the peptide supported by a reduction in tumor microvasculature.