Transtumoral targeting enabled by a novel neuropilin-binding peptide.

We have recently described a class of peptides that improve drug delivery by increasing penetration of drugs into solid tumors. These peptides contain a C-terminal C-end Rule (CendR) sequence motif (R/K)XX(R/K), which is responsible for cell internalization and tissue-penetration activity. Tumor-specific CendR peptides contain both a tumor-homing motif and a cryptic CendR motif that is proteolytically unmasked in tumor tissue. A previously described cyclic tumor-homing peptide, LyP-1 (sequence: CGNKRTRGC), contains a CendR element and is capable of tissue penetration. We use here the truncated form of LyP-1, in which the CendR motif is exposed (CGNKRTR; tLyP-1), and show that both LyP-1 and tLyP-1 internalize into cells through the neuropilin-1-dependent CendR internalization pathway. Moreover, we show that neuropilin-2 also binds tLyP-1 and that this binding equally activates the CendR pathway. Fluorescein-labeled tLyP-1 peptide and tLyP-1-conjugated nanoparticles show robust and selective homing to tumors, penetrating from the blood vessels into the tumor parenchyma. The truncated peptide is more potent in this regard than the parent peptide LyP-1. tLyP-1 furthermore improves extravasation of a co-injected nanoparticle into the tumor tissue. These properties make tLyP-1 a promising tool for targeted delivery of therapeutic and diagnostic agents to breast cancers and perhaps other types of tumors.

Optimization of iron oxide nanoparticle detection using ultrashort echo time pulse sequences: comparison of T1, T2*, and synergistic T1- T2* contrast mechanisms.

Iron oxide nanoparticles (IONPs) are used in various MRI applications as negative contrast agents. A major challenge is to distinguish regions of signal void due to IONPs from those due to low signal tissues or susceptibility artifacts. To overcome this limitation, several positive contrast strategies have been proposed. Relying on IONP T(1) shortening effects to generate positive contrast is a particularly appealing strategy because it should provide additional specificity when associated with the usual negative contrast from effective transverse relaxation time (T(2)*) effects. In this article, ultrashort echo time imaging is shown to be a powerful technique which can take full advantage of both contrast mechanisms. Methods of comparing T(1) and T(2)* contrast efficiency are described and general rules that allow optimizing IONP detection sensitivity are derived. Contrary to conventional wisdom, optimizing T(1) contrast is often a good strategy for imaging IONPs. Under certain conditions, subtraction of a later echo signal from the ultrashort echo time signal not only improves IONP specificity by providing long T(2)* background suppression but also increases detection sensitivity, as it enables a synergistic combination of usually antagonist T(1) and T(2)* contrasts. In vitro experiments support our theory, and a molecular imaging application is demonstrated using tumor-targeted IONPs in vivo.

Vascular homing peptides with cell-penetrating properties.

In vivo screening of phage-displayed peptide libraries has revealed extensive molecular heterogeneity in the blood vessels of individual normal tissues and shown that pathological lesions put their signature on the vasculature. In tumors, both blood and lymphatic vessels differ from normal vessels. Moreover, the molecular changes in the vasculature parallel progression in tumor development, hence making the vessels in premalignant lesions distinguishable from normal vessels and from the vessels in malignant tumors of the same tissue. Some of the tumor-homing peptides penetrate into tumor endothelial cells (and tumor cells), but not into endothelial cells in normal tissues or other normal cells. Thus, these cell-penetrating peptides are cell type-specific. Peptides that home to tumor vasculature have been shown to be useful in directing therapeutic agents to experimental tumors. The cell penetrating peptides may be particularly useful in drug delivery because they can take their payload inside the target cells and even into a specific subcellular organelle such as the nucleus.

Vascular zip codes in angiogenesis and metastasis.

In vivo screening of phage-displayed peptide libraries has revealed extensive molecular differences in the blood vessels of individual normal tissues. Pathological lesions also put their signature on the vasculature; in tumours, both blood and lymphatic vessels differ from normal vessels. The changes that characterize tumour blood vessels include selective expression of certain integrins. Peptides isolated by in vivo phage display for homing to tumours have been shown to be useful in directing therapeutic agents to experimental tumours. The targeting can enhance the efficacy of the therapy while reducing side effects. Phage screening has also revealed lung-specific vascular markers that promote tumour metastasis to the lungs by mediating specific adherence of tumour cells to the lung vasculature. These phage-screening studies have revealed a previously unsuspected degree of vascular specialization and provide potentially useful guidance devices for targeted therapies.

Decreased focal adhesion kinase suppresses papilloma formation during experimental mouse skin carcinogenesis.

Although focal adhesion kinase (FAK) is elevated in epithelial cancers, it is not known whether FAK expression influences tumor development in vivo. We found that fak +/- heterozygous mice display reduced 7,12-dimethylbenz[a]anthracene-induced papilloma formation that correlates with reduced FAK protein expression in the skin. However, the frequency of malignant conversion of papillomas into carcinomas is indistinguishable in fak +/- mice and their wild-type fak +/+ littermates, most likely because papilloma FAK protein expression is elevated to wild-type levels. We also found that keratinocyte FAK protein expression is important for cellular responses downstream of ras in vitro (monitored by extracellular signal-regulated kinase activation after integrin engagement). Because 7,12-dimethylbenz[a]anthracene induces an activating mutation of H-ras, this provides one possible explanation for suppression of papilloma formation when FAK protein is limiting.

Suppression of murine collagen-induced arthritis by targeted apoptosis of synovial neovasculature.

Because angiogenesis plays a major role in the perpetuation of inflammatory arthritis, we explored a method for selectively targeting and destroying new synovial blood vessels. Mice with collagen-induced arthritis were injected intravenously with phage expressing an RGD motif. In addition, the RGD peptide (RGD-4C) was covalently linked to a proapoptotic heptapeptide dimer, D(KLAKLAK)2, and was systemically administered to mice with collagen-induced arthritis. A phage displaying an RGD-containing cyclic peptide (RGD-4C) that binds selectively to the alpha(v)beta3 and alpha(v)beta5 integrins accumulated in inflamed synovium but not in normal synovium. Homing of RGD-4C phage to inflamed synovium was inhibited by co-administration of soluble RGD-4C. Intravenous injections of the RGD-4C-D(KLAKLAK)2 chimeric peptide significantly decreased clinical arthritis and increased apoptosis of synovial blood vessels, whereas treatment with vehicle or uncoupled mixture of the RGD-4C and the untargeted proapoptotic peptide had no effect. Targeted apoptosis of synovial neovasculature can induce apoptosis and suppress clinical arthritis. This form of therapy has potential utility in the treatment of inflammatory arthritis.

A signaling pathway from the alpha5beta1 and alpha(v)beta3 integrins that elevates bcl-2 transcription.

Integrin-mediated cell adhesion is necessary for the survival of many cell types, and loss of adhesion causes apoptosis. We have previously shown that the alpha5beta1 integrin supports cell survival on fibronectin and increases Bcl-2 protein expression. Here we show that bcl-2 transcription is elevated in cells that attach to fibronectin through alpha(v)beta1 or to vitronectin through alpha(v)beta3 but is not elevated in cells attaching through the alpha(v)beta1 integrin. Bcl-2 protein expression and protection from apoptosis under serum-free conditions correlated with bcl-2 transcription. This integrin-mediated regulation of bcl-2 is Shc- and FAK-dependent, and activation of Ras by FAK is required. Furthermore, Ras mediates this up-regulation of bcl-2 by activating the phosphatidylinositol 3-kinase-AKT pathway. Mitogen-activated protein kinase did not appear to be necessary for the activation of bcl-2 transcription. Therefore, our work characterizes the pathway that mediates the effect of integrins on bcl-2 transcription and cell survival.

Solution structures and integrin binding activities of an RGD peptide with two isomers.

The Arg-Gly-Asp (RGD) sequence serves as the primary integrin recognition site in extracellular matrix proteins, and peptides containing this sequence can mimic the activities of the matrix proteins. Depending on the context of the RGD sequence, an RGD-containing peptide may bind to all of the RGD-directed integrins, to a few, or to only a single one. We have previously isolated from a phage-displayed peptide library a cyclic peptide that binds avidly to the alpha(v)beta3 and alpha(v)beta5 integrins but does not bind to other closely related integrins. This peptide, ACDCRGDCFCG, exists in two natural configurations depending on internal disulfide bonding. The peptide with the 1-4; 2-3 disulfide bond arrangement accounts for most of the alpha(v) integrin binding activity, whereas the 1-3; 2-4 peptide is about 10-fold less potent. Solution structure analysis by nuclear magnetic resonance reveals an entirely different presentation of the RGD motif in the two isomers of RGD-4C. These results provide new insight into the ligand recognition specificity of integrins.

A fibronectin fragment inhibits tumor growth, angiogenesis, and metastasis.

We have shown previously that a polymeric form of fibronectin is strongly antimetastatic when administered systemically to tumor-bearing mice. The polymeric fibronectin, sFN, is formed in vitro by treating soluble fibronectin with a 76-aa peptide, III1-C, which is derived from the first type III repeat in fibronectin. Here we show that the III1-C peptide and sFN also reduce tumor growth in mice, and that this effect correlates with a low density of blood vessels in the tumors of the treated mice. III1-C also polymerized fibrinogen, and the fibrinogen polymer, sFBG, had antitumor and antiangiogenic effects similar to those of sFN. Mice that had been injected s.c. with three different types of human tumor cells and treated with biweekly i.p. injections of III1-C, sFN, or sFBG over a 5-week period had tumors that were 50-90% smaller than those of control mice. Blood vessel density in the tumors of the treated mice was reduced by 60-80% at the end of the experiment. Xenograft tumors from a human breast carcinoma line (MDA-MB-435) were particularly susceptible to these treatments. Metastasis into the lungs from the primary s.c. tumors also was inhibited in the mice treated with III1-C and the two polymers. The III1-C peptide is an antiangiogenic and antimetastatic agent. Because of its ability to suppress tumor growth, angiogenesis, and metastasis, we have named the III1-C peptide anastellin [from anastello (Greek), inhibit, force a retreat].

Platelet-derived growth factor receptor beta and vascular endothelial growth factor receptor 2 bind to the beta 3 integrin through its extracellular domain.

Integrin-mediated cell attachment and growth factor stimulation often act synergistically on cell proliferation, differentiation, migration, and survival. Some of these synergistic effects depend on the physical interaction of integrins with growth factor receptors. Here we examine the nature of the physical interaction between the alpha(v)beta(3) integrin and two receptor tyrosine kinases (RTKs), the platelet-derived growth factor receptor beta (PDGF-Rbeta) and the vascular endothelial growth factor receptor 2 (VEGF-R2, also known as KDR and flk-1). Both of these RTKs associate with the alpha(v)beta(3) integrin but do not associate with beta(1) integrins. Furthermore, growth factor stimulation of these RTKs promotes increased cell proliferation and migration when cells are attached to the alpha(v)beta(3) ligand, vitronectin. We show that alpha(v)beta(3) in which the beta(3) cytoplasmic domain is deleted or replaced with the beta(1) cytoplasmic domain coimmunoprecipitates with PDGF-Rbeta and VEGF-R2. The beta(3) extracellular domain alone was sufficient for the PDGF-Rbeta association whereas the VEGF-R2 association required the presence of the alpha(v) subunit. Activation of the RTKs by their ligands was not required for them to associate with the integrin. Cell migration to PDGF was enhanced in the cells transfected with the chimeric subunit containing the beta(3) extracellular domain but not when that domain came from the beta(1) subunit. These results show that the interactions that lead to the association of the alpha(v)beta(3) integrin with PDGF-Rbeta and VEGF-R2 and enhancement of RTK activity take place outside the cell.

An address system in the vasculature of normal tissues and tumors.

The vasculature of individual tissues is highly specialized. The endothelium in lymphoid tissues expresses tissue-specific receptors for lymphocyte homing, and recent work utilizing phage homing has revealed an unprecedented degree of specialization in the vasculature of other normal tissues. In vivo screening of libraries of phage that displace random peptide sequences on their surfaces has yielded specific homing peptides for a large number of normal tissues. The tissue-specific endothelial molecules to which the phage peptides home may serve as receptors for metastasizing malignant cells. Probing of tumor vasculature has yielded peptides that home to endothelial receptors expressed selectively in angiogenic neovasculature. These receptors, and those specific for the vasculature of individual normal tissues, are likely to be useful in targeting therapies to specific sites.

Superfibronectin, a multimeric form of fibronectin, increases HIV infection of primary CD4+ T lymphocytes.

The ability of viruses and bacteria to interact with the extracellular matrix plays an important role in their infectivity and pathogenicity. Fibronectin is a major component of the extracellular matrix in lymph node tissue, the main site of HIV deposition and replication during the chronic phase of infection. Therefore, we asked whether matrix fibronectin (FN) could affect the ability of HIV to infect lymphocytes. To study the role of matrix FN on HIV infection, we used superfibronectin (sFN), a multimeric form of FN that closely resembles in vivo matrix FN. In this study we show that HIV-1IIIB efficiently binds to multimeric fibronectin (sFN) and that HIV infection of primary CD4+ lymphocytes is enhanced by >1 order of magnitude in the presence of sFN. This increase appears to be due to increased adhesion of viral particles to the cell surface in the presence of sFN, followed by internalization of virus. Enzymatic removal of cell surface proteoglycans inhibited the adhesion of HIV-1IIIB/sFN complexes to lymphocytes. In contrast, Abs to integrins had no effect on binding of HIV-1IIIB/sFN complexes to lymphocytes. The III1-C peptide alone also bound HIV-1IIIB efficiently and enhanced HIV infection, although not as effectively as sFN. HIV-1IIIB gp120 envelope protein binds to the III1-C region of sFN and may be important in the interaction of virus with matrix FN. We conclude that HIV-1IIIB specifically interacts with the III1-C region within matrix FN, and that this interaction may play a role in facilitating HIV infection in vivo, particularly in lymph node tissue.

Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis.

Phage that display a surface peptide with the NGR sequence motif home selectively to tumor vasculature in vivo. A drug coupled to an NGR peptide has more potent antitumor effects than the free drug [W. Arap et al., Science (Washington DC), 279: 377-380, 1998]. We show here that the receptor for the NGR peptides in tumor vasculature is aminopeptidase N (APN; also called CD13). NGR phage specifically bound to immunocaptured APN and to cells engineered to express APN on their surface. Antibodies against APN inhibited in vivo tumor homing by the NGR phage. Immunohistochemical staining showed that APN expression is up-regulated in endothelial cells within mouse and human tumors. In another tissue that undergoes angiogenesis, corpus luteum, blood vessels also expressed APN, but APN was not detected in blood vessels of various other normal tissues stained under the same conditions. APN antagonists specifically inhibited angiogenesis in chorioallantoic membranes and in the retina and suppressed tumor growth. Thus, APN is involved in angiogenesis and can serve as a target for delivering drugs into tumors and for inhibiting angiogenesis.

A peptide mimic of E-selectin ligand inhibits sialyl Lewis X-dependent lung colonization of tumor cells.

Selectins bind to carbohydrate ligands in a calcium-dependent manner and play critical roles in host defense and possibly in tumor metastasis. To isolate peptides that mimic E-selectin ligands, we screened a phage peptide library using E-selectin as a target molecule. This attempt unexpectedly failed, probably because the binding affinity of E-selectin to its ligand is low. We then took an approach that is analogous to the isolation of anti-idiotype antibodies and were able to isolate peptides that bound to anticarbohydrate antibodies recognizing E-selectin ligands. These peptides, enriched for their binding to anti-Lewis A antibody, were found to bind to E-, P- and L-selectins in a calcium-dependent manner. Phage harboring the identified peptide IELLQAR and synthetic peptides having the same sequence inhibited the binding of sialyl Lewis X or sialyl Lewis A oligosaccharides to E-selectin. The adhesion of HL-60 and B16 melanoma cells expressing sialyl Lewis X to E-selectin was also inhibited by the phage-displaying IELLQAR peptide. Moreover, i.v. injected IELLQAR peptide inhibited the lung colonization of mouse B16 melanoma and human lung tumor cells expressing sialyl Lewis X. These results demonstrate that it is possible to isolate peptides mimicking carbohydrate ligands by screening the peptides for binding to anticarbohydrate antibodies and then using them to inhibit carbohydrate-dependent experimental tumor metastasis.

Integrin alpha(2)beta(1) (VLA-2) is a principal receptor used by neutrophils for locomotion in extravascular tissue.

Cell adhesion molecules are critically involved in the multistep process of leukocyte recruitment in inflammation. The specific receptors used by polymorphonuclear leukocytes (PMN) for locomotion in extravascular tissue have as yet not been identified. By means of immunofluorescence flow cytometry and laser scanning confocal microscopy, this study demonstrated that surface expression of the alpha(2)beta(1) (VLA-2) integrin, though absent on blood PMN, is induced in extravasated PMN collected from human skin blister chambers, and rat PMN accumulated in the peritoneal cavity after chemotactic stimulation. Intravital time-lapse videomicroscopy was used to investigate chemoattractant-induced PMN locomotion in the rat mesentery in vivo. Local administration of function-blocking monoclonal antibody or peptide recognizing the alpha(2)beta(1) integrin reduced PMN migration velocity in the extravascular tissue by 73% +/- 3% and 70% +/- 10%, respectively (means +/- SD). The distance f-met-leu-phe peptide (fMLP)-stimulated human PMN migrated in a collagen gel in vitro was markedly reduced by treatment with anti-alpha(2) mAbs or peptide, whereas no effect was observed with antibodies or peptides recognizing the alpha(4)beta(1) or alpha(5)beta(1) integrins. Further evidence for a critical role of expression of alpha(2)beta(1) integrin in PMN locomotion in extravascular tissue was obtained in the mouse air pouch model of acute inflammation where chemoattractant-induced PMN recruitment was substantially inhibited by local anti-alpha(2) mAb treatment. Thus, expression of alpha(2)beta(1) integrin on extravasated PMN has been identified and a novel role of this receptor in regulating the extravascular phase of leukocyte trafficking in inflammation has been formulated. (Blood. 2000;95:1804-1809)

R-Ras contains a proline-rich site that binds to SH3 domains and is required for integrin activation by R-Ras.

R-Ras contains a proline-rich motif that resembles SH3 domain-binding sites but that has escaped notice previously. We show here that this site in R-Ras is capable of binding SH3 domains and that the SH3 domain binding may be important for R-Ras function. A fusion protein containing the SH3 domains of the adaptor protein Nck interacted strongly with the R-Ras proline-rich sequence and with the intact protein. The binding was independent of whether R-Ras was in its GDP or GTP form. The Nck binding, which was mediated by the second of the three SH3 domains of Nck, was obliterated by mutations in the proline-rich sequence of R-Ras. The interaction of Nck with R-Ras could also be shown in yeast two-hybrid assays and by co-immunoprecipitation in human cells transfected with Nck and R-Ras. Previous results have shown that the expression of a constitutively active R-Ras mutant, R-Ras(38V), converts mouse 32D monocytic cells into highly adherent cells. Introducing the proline mutations into R-Ras(38V) suppressed the effect of R-Ras on 32D cell adhesion while not affecting GTP binding. These results reveal an unexpected regulatory pathway that controls R-Ras through an SH3 domain interaction. This pathway appears to be important for the ability of R-Ras to control cell adhesion.

Replacing two conserved tyrosines of the EphB2 receptor with glutamic acid prevents binding of SH2 domains without abrogating kinase activity and biological responses.

Eph receptor tyrosine kinases play key roles in pattern formation during embryonic development, but little is known about the mechanisms by which they elicit specific biological responses in cells. Here, we investigate the role of tyrosines 605 and 611 in the juxtamembrane region of EphB2, because they are conserved Eph receptor autophosphorylation sites and demonstrated binding sites for the SH2 domains of multiple signaling proteins. Mutation of tyrosines 605 and 611 to phenylalanine impaired EphB2 kinase activity, complicating analysis of their function as SH2 domain binding sites and their contribution to EphB2-mediated signaling. In contrast, mutation to the negatively charged glutamic acid disrupted SH2 domain binding without reducing EphB2 kinase activity. By using a panel of EphB2 mutants, we found that kinase activity is required for the changes in cell-matrix and cell - cell adhesion, cytoskeletal organization, and activation of mitogen-activated protein (MAP) kinases elicited by EphB2 in transiently transfected cells. Instead, the two juxtamembrane SH2 domain binding sites were dispensable for these effects. These results suggest that phosphorylation of tyrosines 605 and 611 is critical for EphB2-mediated cellular responses because it regulates EphB2 kinase activity.

Targeting tumor vasculature with homing peptides from phage display.

Tumor vasculature expresses a number of molecular markers at much lower levels than those seen in the blood vessels of normal tissues, and in some cases, such markers are undetectable. The presence of these markers relates to angiogenesis; the same markers are shared by all blood vessels undergoing angiogenesis. The endothelial cells, pericytes and smooth muscle cells, and the vascular extracellular matrix in angiogenic vessels can each express such markers. Molecularly, they represent vascular growth factor receptors, cell adhesion proteins and their receptors. Screening of phage display libraries for peptides that home to tumor vasculature when injected into mice has recently provided a new tool for analyzing the distinguishing features of tumor vasculature. Tumor-homing peptides isolated in this manner, as well as an antibody against a form of fibronectin expressed in tumor blood vessels, have been found to serve as targeting devices to concentrate drugs and other therapeutic materials to tumors in in vivo models. Such a targeting strategy can therefore potentially improve the efficacy of drugs and reduce their side effects.

An Eph receptor regulates integrin activity through R-Ras.

The ability of integrins to mediate cell attachment to extracellular matrices and to blood proteins is regulated from inside the cell. Increased ligand-binding activity of integrins is critical for platelet aggregation upon blood clotting and for leukocyte extravasation to inflamed tissues. Decreased adhesion is thought to promote tumor cell invasion. R-Ras, a small intracellular GTPase, regulates the binding of integrins to their ligands outside the cell. Here we show that the Eph receptor tyrosine kinase, EphB2, can control integrin activity through R-Ras. Cells in which EphB2 is activated become poorly adherent to substrates coated with integrin ligands, and a tyrosine residue in the R-Ras effector domain is phosphorylated. The R-Ras phosphorylation and loss of cell adhesion are causally related, because forced expression of an R-Ras variant resistant to phosphorylation at the critical site made cells unresponsive to the anti-adhesive effect of EphB2. This is an unusual regulatory pathway among the small GTPases. Reduced adhesiveness induced through the Eph/R-Ras pathway may explain the repulsive effect of the Eph receptors in axonal pathfinding and may facilitate tumor cell invasion and angiogenesis.