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Therapeutics, Targets, and Chemical Biology

De Novo Design of a Tumor-Penetrating Peptide

Luca Alberici1,2,3,4, Lise Roth5, Kazuki N. Sugahara1,2, Lilach Agemy1,2, Venkata R. Kotamraju1,2,Tambet Teesalu1,2, Claudio Bordignon3,4, Catia Traversari3, Gian-Paolo Rizzardi3, and Erkki Ruoslahti1,2

AbstractPoor penetration of antitumor drugs into the extravascular tumor tissue is often a major factor limiting the

efficacy of cancer treatments. Our group has recently described a strategy to enhance tumor penetration ofchemotherapeutic drugs through use of iRGD peptide (CRGDK/RGPDC). This peptide comprises two sequencemotifs: RGD, which binds to avb3/5 integrins on tumor endothelia and tumor cells, and a cryptic CendR motif(R/KXXR/K-OH). Once integrin binding has brought iRGD to the tumor, the peptide is proteolytically cleavedto expose the cryptic CendR motif. The truncated peptide loses affinity for its primary receptor and binds toneuropilin-1, activating a tissue penetration pathway that delivers the peptide along with attached or co-administered payload into the tumor mass. Here, we describe the design of a new tumor-penetrating peptidebased on the current knowledge of homing sequences and internalizing receptors. The tumor-homing motif inthe new peptide is the NGR sequence, which binds to endothelial CD13. The NGR sequence was placed in thecontext of a CendR motif (RNGR), and this sequence was embedded in the iRGD framework. The resultingpeptide (CRNGRGPDC, iNGR) homed to tumor vessels and penetrated into tumor tissue more effectively thanthe standard NGR peptide. iNGR induced greater tumor penetration of coupled nanoparticles and co-admin-istered compounds than NGR. Doxorubicin given together with iNGR was significantly more efficacious than thedrug alone. These results show that a tumor-specific, tissue-penetrating peptide can be constructed from knownsequence elements. This principle may be useful in designing tissue-penetrating peptides for other diseases.Cancer Res; 73(2); 804–12. �2012 AACR.

IntroductionThe vasculature of each tissue is unique in terms of

protein expression and these molecular differences arereferred to as "vascular zip codes" (1). The selectivelyexpressed proteins provide targets for specific delivery ofdiagnostic and therapeutic compounds to the vasculature ofdesired tissues. Currently, a variety of tumor-targeting pep-tides are in preclinical and clinical development. However,vascular abnormalities, fibrosis, and contraction of extra-cellular matrix contribute to an increased interstitial fluidpressure inside the tumor, which impedes drug delivery intothe extravascular tumor tissue (2).

Our group has recently reported the identification of theCendR motif (R/KXXR/K) that is capable of increasing the

penetration of peptides, chemicals, and synthetic and biologicnanoparticles into tissues through the engagement of neuro-pilin-1 (NRP-1; ref. 3). Specific penetration into tumors wasachieved through the use of an iRGD peptide (CRGDK/RGPDC;ref. 4). iRGD, identified by in vivo phage display for tumor-homing peptides, combines targeting to tumor vessels andtumor parenchyma through an RGD motif with the cell-inter-nalizing and tissue-penetrating properties of a CendR motifRGDK/R in the peptide (4). iRGDmechanism of action involves3 steps. First, the RGD sequence binds to avb3/5 integrins.Then, a proteolytic cleavage by a yet-to-be-identified host pro-tease(s) exposes the CendR motif, which is now able to inter-act with NRP-1 to trigger the internalization process. Thisstrategy allows the activation of the CendR motif only in a tar-geted tissue, avoiding NRP-1 activation in normal vasculature.Interestingly, iRGD triggers a specific tumor penetration of notonly iRGD-coupled compounds but also drugs co-administeredwith free iRGD peptide (5). The CendR motif also activates thepenetration pathway through binding to NRP-2 (6).

Potentially, the addition of a cryptic CendR motif couldincrease the penetration of other tumor targeting peptides,providing more tools to overcome the poor delivery of drugsto tumors. We set out to test this hypothesis using the NGRtumor-homing motif. The NGR sequence was identified byin vivo phage display in tumor-bearing mice (7). Initially, itwas thought to bind one or more of the integrins selectivelyexpressed in angiogenic vessels (7, 8). This idea was furthersupported by the discovery that the asparagine in the NGR

Authors' Affiliations: 1Cancer Center, Sanford-Burnham Medical Re-search Institute, La Jolla; 2Center for Nanomedicine, University of Califor-nia, Santa Barbara, California; 3MolMed S.p.A.; 4Universit�a Vita-Salute SanRaffaele, Milano, Italy; and 5Division of Vascular Oncology andMetastasis,German Cancer Research Center (DKFZ), Heidelberg, Germany

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Erkki Ruoslahti, Cancer Center, Sanford-Burnham Medical Research Institute, 10901 N. Torrey Pines Rd.,La Jolla, CA 92037. Phone: 858-795-5023; Fax: 858-795-5323; E-mail:[email protected]

doi: 10.1158/0008-5472.CAN-12-1668

�2012 American Association for Cancer Research.

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motif undergoes a spontaneous deamidation reaction thatyields iso-aspartic acid (isoDGR), generating an RGD mimetic(9, 10). However, the unaltered NGR motif also specificallyhomes to tumor vessels, where it binds to an isoform ofaminopeptidase N (CD13; refs. 11, 12). NGR peptides havebeen used to target a variety of agents into tumors; an NGRconjugate of human TNF-a is in advanced clinical trials forcancer therapy (13–16). Here, we combined theNGRmotif witha CendR motif to create a new tumor-homing peptide withtissue-penetrating properties.

Materials and MethodsAnimal useAll procedures on the animals, including those to ensure

minimizing discomfort, have been carried out according to theprotocol approved at the Sanford-Burnham Medical ResearchInstitute (La Jolla, CA).

Preparation of compoundsSynthetic peptides (4), peptide-coated nanoworms (NW;

ref. 17), and peptide-expressing T7 phage (18) were preparedas described elsewhere. Doxorubicin was purchased fromSigma-Aldrich. Evans Blue was purchased from MP Bio-medicals.

Cell lines and tumor modelsHuman umbilical vein endothelial cells (HUVEC; Lonza)

were cultured in complete EGM-2 medium from Lonza. 4T1cells were cultured in Dulbecco's Modified Eagle Medium(DMEM; Thermo Scientific) supplemented with 10% FBS andpenicillin/streptomycin (Gibco). All tumor cell lines werebought and authenticated by American Type Culture Collec-tion. Orthotopic 4T1 breast tumorswere generated by injecting105 cells into the mammary fat pad of female BALB/c mice atthe age of 4 to 6 weeks (Harlan Sprague-Dawley).

In vitro phage-binding and internalization assaysPhage amplification, purification, titration, sequencing, and

UV inactivation were conducted as reviewed (18). One millioncells were incubated with 1010 plaque-forming units (pfu) ofpurified phage in DMEM/1%BSA at 4�C for binding or 37�C forinternalization. The cells were washed with cold DMEM/BSA 4times, lysed in lysogeny broth (LB) containing 1%Nonidet P-40(LB-NP40), and titrated. In internalization assays, the secondwash was replaced with an acid wash (500 mmol/L NaCl, 0.1mol/L glycine, 1% BSA, pH 2.5) to remove and inactivate phagebound to the cell surface. In inhibition assays, the cells wereincubated with 1 mg/mL of neutralizing anti-NRP-1 antibody(R&D Systems), control IgG (Santa Cruz Biotechnology), or 10-fold excess of UV-inactivated phage 15 minutes before addingthe phage of interest.

Ex vivo tumor-dipping assaysThe assays were conducted as described elsewhere (5, 6).

Briefly, 4T1 tumor–bearing mice were anesthetized and per-fused through the heart with PBS containing 1% BSA. Thetumors were excised and incubated with 109 pfu of phagein DMEM/1% BSA for 1 hour at 37�C. After extensive washes

with PBS, the tumors were lysed in 1 mL of LB/NP-40 forphage titration. In some cases, the tumors were fixed in PBScontaining 4% paraformaldehyde (PFA) and processed forimmunostaining.

Biodistribution of peptides, phage, and nanowormsIn vivo tumor-homing experiments with peptides, phage,

and nanoworms were conducted as described (4, 17). Briefly,109 pfu of phage particles were injected into the tail vein oftumor-bearing mice and allowed to circulate for 40 minutes.Themice were perfused through the heart with PBS containing1% BSA under deep anesthesia. The tumors and tissues wereexcised and mechanically homogenized for phage titrationor fixed in 4% PFA for immunostaining. In some cases, 50 mgof anti-NRP-1 antibody or control IgG was intravenouslyinjected 15 minutes before the phage injection. For the pep-tide-homing studies, 100mLof 1mmol/L FAM-labeled peptideswere intravenously injected into tumor mice and allowed tocirculate for 1 hour. In case of the nanoworms, particles ata dose of 5 mg/kg of iron were injected into the tail vein oftumor mice and allowed to circulate for 5 hours. After perfu-sion of the mice, tissues were collected, macroscopicallyobserved under UV light (Illuminatool Bright Light SystemLT-9900), and processed for immunostaining. Intratumoralaccumulation of nanoworms was quantified with ImageJ.

ImmunofluorescenceImmunofluorescence on cells was conducted using

10 mmol/L FAM peptides and following the protocol previ-ously described (19). Immunofluorescence on frozen sec-tions was conducted as described earlier (4) using thefollowing antibodies at 1:200 dilution: rat anti-mouse CD31Alexa-594 (Invitrogen), rabbit anti-T7 phage (3), and donkeyanti-rabbit Alexa 488 (Invitrogen). Images were taken using aFluoview confocal microscope (Olympus).

In vivo systemic permeability assayTumor-bearing mice were injected intravenously with

4 mmol/kg of peptide combined with 1 mg of Evans Blue,10mg/kg of free doxorubicin, or 5mg iron/kg of CGKRK-coatednanoworms (20). After indicated time of circulation, the micewere perfused through the heart with PBS supplemented with1% BSA, and tissues were collected. Evans Blue was extractedby incubating the tissues in 1 mL of 2,2 N-methylformamideovernight at 37�C with mild shaking. After centrifugation, theoptical density (OD)600 of the supernatant was measured. Toassess the extravasation of CGKRK-NWs, tissues were fixed in4% PFA overnight at 4�C and subjected to immunostainingfor CD31-positive blood vessels. For doxorubicin quantification(21), excised tissues weremechanically homogenized in 1mL of1% SDS containing 1 mmol/L H2SO4 and frozen overnight in2 mL of chloroform/isopropyl alcohol (1:1, v/v). The sampleswere melted, vortexed, and centrifuged at 16,000 � g for 15minutes, and OD490 of the organic (lower) phase was measured.

Tumor treatment studiesMice bearing orthotopic 4T1 breast tumors at 50 mm3

received intravenous injections of free doxorubicin (3 mg/kg)


www.aacrjournals.org Cancer Res; 73(2) January 15, 2013 805




or PBS, combined with 4 mmol/kg of peptide every other day.Tumor growth and body weight were monitored every otherday. The tumor volume was calculated using the followingformula: volume (mm3) ¼ (d2 � D)/2, where d is the smallestand D is the largest tumor diameters.

Statistical analysisAll data were analyzed with one-way ANOVA. Tumor treat-

ment studies were analyzed with 2-way ANOVA for repeatedmeasurements.

ResultsDesign of iNGR peptide

We used 3 elements to create the iNGR peptide(CRNGRGPDC): the NGR motif, a CendR motif (RNGR) over-lapping with the NGR motif, and a cleavable consensus (GPD)from the iRGD peptide. The cyclic conformation requiredfor high affinity binding of NGR to CD13 (22) was obtainedthrough the addition of cysteines at the N- and C- terminus ofthe peptide. We also prepared the truncated version of iNGR

that is expected to result from proteolytic activation of iNGR(CRNGR), which we refer to as iNGRt. The conventional NGR(CNGRC), RGD (CRGDC), iRGD (CRGDKGDPC), and activatediRGD (CRGDK) peptides were used as controls. The peptidesused in this study are summarized in Supplementary Table S1.

iNGR and NGR bind to the same primary receptorHUVECs) express on their surface both the NGR receptor

CD13 and the CendR receptor NRP-1 (Supplementary Fig. S1).iNGR bound to HUVECs as efficiently as CNGRC, whethertested as FAM-labeled peptide (Fig. 1A) or displayed on phage(Fig. 1B, black columns). As expected, the iNGR phage did notbind CD13þ U937 monocytes (Supplementary Fig. S2A–S2C),as previously reported for the CNGRC peptide (11). UV-inactivated phage displaying CNGRC inhibited the HUVECbinding of the iNGR phage in a dose-dependent fashion,whereas inactivated CRGDC did not differ from insertlesscontrol phage in this regard (Fig. 1C). CNGRC phage did notinhibit the binding of iRGD phage to HUVECs (Supplemen-tary Fig. S3A). Moreover, only in vitro deamidated CNGRCand iNGR phage showed binding to immobilized avb3

Figure 1. iNGR shares the samereceptor with CNGRC and, uponactivation, strongly interacts withNRPs. A, HUVECs were incubatedfor 2hours at 4�Cwith FAM-labeledARA (ARALPSQRSR; ref. 28),CNGRC, iNGR, or iNGRt peptides.Cells were fixed and imaged with aFluoview confocal microscope.Scale bars, 100 mm. B and D, ten-fold excess of UV-inactivatedRPARPARor RPARPARA phage or1 mg/mL of control or neutralizingNRP-1 antibodywas used to inhibitthe binding (B) or the internalization(D) of CNGRC, iNGR, or iNGRtphage in HUVECs. The results areshown as fold increase overinsertless phage. �, P < 0.05; one-way ANOVA. Error bars, SE. C,dose-dependent inhibition of iNGRphage binding to HUVECs by UV-inactivated phage. iNGR phagebinding without inhibitors wasconsidered as 100%. Error bars,SE. E and F, dose-dependentbinding of phage to purified NRP-1(E) or NRP-2 (F) proteins. Thenumber of phage bound to theproteins was quantified using acombination of a rabbit anti-T7phage antibody and a horseradishperoxidase (HRP)-labeled goatanti-rabbit antibody.

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Cancer Res; 73(2) January 15, 2013 Cancer Research806




integrin (Supplementary Fig. S3B). These results suggest thatCNGRC and iNGR bind to the same primary receptor throughthe NGR motif and that the conversion of asparagine toaspartate does not take place during phage production,purification, storage, or during the incubations. Therefore,the binding of CNGRC and iNGR to cells is not due to isoDGRinteracting with avb3/5 integrins.

The iNGRCendRmotif interactswithNRPs andpromotescell internalizationWe constructed phage displaying a truncated version

of iNGR in which the CRNGR CendR motif occupies aC-terminal position (iNGRt). This phage bound avidly toHUVECs, likely due to an interaction with NRP-1 (Fig. 1A andB). Indeed, iNGRt phage binding to HUVECs was reducedby preincubation with a UV-inactivated phage expressing aprototypic CendR motif peptide (RPARPAR) or a neutraliz-ing NRP-1 antibody (Fig. 1B), indicating involvement of theCendR/NRP-1 pathway. Preincubation with a phage display-ing a peptide with a blocked CendR motif (RPARPARA),and a control antibody, had no effect on iNGRt binding toHUVECs. The binding of intact iNGR was not affected bythe UV-inactivated RPARPAR phage or NRP-1 antibodyshowing that NRP-1 is not involved in the initial bindingof the peptide. Measuring phage internalization by incubat-ing phage with HUVECs at 37�C, followed by a wash with alow pH buffer to inactivate extracellular phage, showedstronger internalization of iNGR and iNGRt than CNGRC(Fig. 1D). Inhibition experiments showed that the internal-ization was mediated by the interaction of the CendR motifwith NRP-1. These results suggest that HUVECs express aprotease capable of activating the cryptic CendR motifembedded in the iNGR peptide. Indeed, mass spectrometryshowed that upon incubation of HUVECs with FAM-iNGRpeptide, only the cleaved FAM-CRNGR fragment (m/z:1076.527) but not the full-length peptide (m/z: 1445.065),was present inside the cells (Supplementary Fig. S4). FAM-CNGRC peptide (m/z: 1020.020) did not penetrate into thecells. Direct proof that the CendR motif within the iNGRpeptide is capable of binding to NRPs was provided byCRNGR phage binding to immobilized NRP-1 (Fig. 1E). Thisphage also bound to NRP-2 (Fig. 1F). In agreement with thefinding that the motif R/KXXR/K has to be in a C-terminalposition to bind to NRPs (the Cend-Rule; ref. 3), only phageexpressing the iNGRt, and the analogous iRGDt, bound tothe NRPs, whereas the corresponding full-length peptidesshowed only background binding. Interestingly, iNGRtshowed higher affinity for NRP-1 and NRP-2 than iRGDt,suggesting that a C-terminal arginine residue (CRNGR)provides higher affinity than lysine (CRGDK).

iNGR penetrates deeper into tumors than NGRWe have previously shown in ex vivo tumor penetration

assays that iRGD uses a CendR-mediated active transportsystem to cross tumor barriers (5). To investigate the iNGR-mediated penetration pathway, we conducted an ex vivo tumorpenetration assay of phage using explants of orthotopic 4T1murine breast tumors (positive for expression of CD13 and

NRP1/2; Fig. 2A). To evaluate the extent of tumor penetration,we titrated the phage recovered from tumors (Fig. 2B) anddetermined the distribution of phage immunoreactivity(Fig. 2C). The CendR-containing phage (iNGR and iRGD)penetrated into the explants with similar patterns, whereasnon-CendR phage (CNGRC and control) remained at the

Figure 2. iNGR penetrates deeper to tumors than NGR. A, expression ofCD13, NRP-1, and NRP-2 on 4T1 cells analyzed by flow cytometry. Theprofiles represent the values of cells stained with appropriate antibodies(solid lines) or an isotype control (shaded). B, explanted 4T1 tumors wereincubated with insertless, CNGRC, iNGR, or iRGD phage. Phage boundto the tumor surface was removed with acid wash, and the number ofphage particles that penetrated into the tumors was quantified by phagetitration. Results are shown as fold increase over insertless phage. Eachvaluewas normalized against tumorweight. �,P < 0.05; one-way ANOVA.Error bars, SE. C, tumor-dipping assays were conducted as in B, andfrozen tumor sections were stained with an anti-T7 phage antibody(green) and DAPI (blue). Scale bars, 100 mm. DAPI, 40,6-diamidino-2-phenylindole.






outer rim of the explants. These results suggest that iNGRand iRGD share a similar CendR-mediated transport mecha-nism to penetrate tumor tissue.

Systemic iNGR selectively accumulates and penetratesinto tumors

Having determined that the CendR motif within the iNGRpeptide can be proteolytically activated to trigger interactionwith NRPs and penetration into cells and tissues, we assess-ed the homing of iNGR in vivo. Intravenously administerediNGR phage accumulated within the tumor and penetratedinto the tumor stromamore than CNGRC phage (Fig. 3A and B,top). The iNGRt phage also showed high tumor penetration,

presumably because of the high expression of NRP-1 on tumorvasculature and tumor cells, but this phage also accumulatedin lungs and heart of the tumor mice. iNGR penetrationcould be blocked by concomitantly administering a neutral-izing anti-NRP-1 antibody, but not a control antibody (Fig. 3B,bottom). Vascular targeting of iNGR was not inhibited bythe anti-NRP-1 treatment (arrows, Fig. 3B, left bottom), sup-porting the notion that the CendR activation occurs after iNGRaccomplishes NGR-dependent vascular targeting. Intrave-nously injected FAM-iNGR peptide also accumulated in 4T1breast tumors (Fig. 3C) and BxPC-3 pancreatic tumors (Sup-plementary Fig. S5A) more strongly than FAM-CNGRC. FAM-iNGR extravasation within tumor tissue was greater than

Figure 3. Systemic iNGR selectivelyaccumulates in and penetrates intotumors. A and B, in vivo phagehoming to orthotopic 4T1 tumors.Phage were intravenously injectedinto 4T1 bearing mice and allowedto circulate for 40 minutes. Afterperfusion of the mice, tissues werecollected and homogenized forphage titration (A) or processed forphage (green) and CD31 (red)immunostaining (B). Bluerepresents DAPI staining. DAPI,40,6-diamidino-2-phenylindole. Insome cases, iNGR phage wasco-injected with 50 mg ofneutralizing NRP-1 antibody orrabbit IgG (B, bottom). �, P < 0.05;one-way ANOVA. Scale bars, 50mm. Error bars, SE. C–E, in vivopeptidehoming to4T1 tumors.Onehundred micrograms of FAM-peptides (green) wereintravenously injected into4T1-bearing mice. One hour later,the mice were perfused, andtissues were collected and imagedon a UV light table (C and E). Then,the tissues were processed forCD31 (red) and nuclei (blue)staining (D). Scale bars, 50 mm. F, invivo homing of NWs to 4T1 tumors.CNGRC- or iNGR-coated NWs(green) were injected into the tailvein of 4T1 tumor mice. After 4hours, themicewere perfused, andtumors were collected andsubjected to CD31 staining (red).Blue represents DAPI staining.Confocal images at �20 and�40 magnifications are shown.Scale bars, 100 mm (�20), 50 mm(�40). The arrows point to bloodvessels positive for phage (B) orpeptide (D). Note that the iNGRphage, peptide, and NWseffectively penetrated 4T1 tumorsand that the anti-NRP-1 antibodyinhibited the tumor penetration ofiNGR phage.

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that of FAM-CNGRC (Fig. 3D). FAM-iNGR selectively pene-trated into tumors and not into control organs (Fig. 3E).Elongated iron oxide nanoparticles (nanoworms) coated withiNGR also showed higher extravasation than CNGRC-NWs(Fig. 3F and Supplementary Fig. S5B). The nanoworms wereless efficient than phage in penetrating the tissue, likelybecause they are larger in size (nanoworms, 30 � 70/200 nm;phage, 55 nm).

iNGR triggers tumor-specific penetration of co-administered compoundsThe engagement of NRP-1 increases vascular permeability

(23), and iRGD triggers this phenomenon specifically in tumors(5). We found that iNGR significantly increased extravasationand accumulation of the albumin-binding dye Evans blue in4T1 tumors, but not in nontumor tissues. CNGRC or vehiclealone had no effect on the biodistribution of the dye (Fig. 4Aand B and Supplementary Fig. S6). iNGR facilitated tumor-specific accumulation of Evans blue in CT26 colon and LLClung tumor models as well (Supplementary Fig. S7). We alsoco-administered iNGR with nanoworms coated with a tumor-homing peptide, CGKRK (20), which brings the nanoworms totumor vessels but does not trigger extravasation. iNGR allowed

the NWs to extravasate into the tumor parenchyma (Fig. 4C).Finally, iNGR triggered more penetration of doxorubicin intothe tumors than doxorubicin alone or doxorubicin combinedwith CNGRC (Fig. 4D).

iNGR enhances anticancer drug efficacyHaving found that iNGR co-administration increased the

local accumulation of doxorubicin within tumors, we investi-gated the effect of iNGR on the activity of doxorubicin. Wetreated orthotopic 4T1 breast tumor mice with a combinationof doxorubicin (3 mg/kg) and 4 mmol/kg of iNGR, a controlpeptide, or PBS every other day. As shown in Fig. 5A, iNGR,but not CNGRC, enhanced the antitumor effect of doxorubi-cin. iNGR alone had no effect on tumor growth. Loss of bodyweight as an indicator of doxorubicin toxicity was not affectedby the peptide co-administration (Fig. 5B). These results showthe potential of iNGR as an adjuvant to increase the efficacy ofco-administered anticancer drugs.

DiscussionWe report here the design of a new tumor-penetrating

peptide, iNGR. The peptide was constructed by combining the

Figure 4. iNGR triggers tumor-specific penetration of co-administered compounds. A and B, one milligram of Evans Blue dye was intravenouslyco-injected with 4 mmol/kg of peptides or PBS. After 40 minutes of circulation, the mice were extensively perfused, and tumors were collected forimaging under white light (A). Evans blue was extracted from the collected tumors and organs and quantified by OD600 measurement (B). C, five mg/kgof FAM-CGKRK–conjugated NWs (green) were injected with or without 4 mmol/kg of iNGR peptide into the tail vein of 4T1 bearing mice. After 5 hours ofcirculation, tumors were collected for CD31 immunostaining (red). Blue represents DAPI staining. DAPI, 40,6-diamidino-2-phenylindole. Tworepresentative images of 3 tumors are shown. Scale bars, 100 mm. D, about 10 mg/kg of doxorubicin (DOX) was intravenously co-injected with4 mmol/kg of the indicated peptides in 4T1-bearing mice. After 1 hour of circulation, the mice were extensively perfused, and the tissues were collectedfor DOX quantification. Results are shown as fold increase over DOX alone. �, P < 0.05; one-way ANOVA. Error bars, SE.






tumor-targeting motif NGR and tissue-penetrating CendRmotif into a 9-amino acid cyclic peptide. The iNGR peptide,homed to tumor vessels, exited the vessels, and penetrated intothe tumor mass. It was able to take both coupled and co-administered payloads with it. When the co-administeredpayload was a drug (doxorubicin), the efficacy of the drugincreased. These results show that it is possible to use theexisting knowledge to construct a new tumor-specific, tissue-penetrating peptide.

The mechanisms underlying iNGR activity are similar tothose described for iRGD (4, 5). The receptor for the tumor-targetingmotif NGR is a variant formof aminopeptidaseN (12).The binding of iNGR to cultured cells was specifically inhibitedby CNGRC, indicating that iNGR binds to the same receptor.NGR peptides are known to spontaneously undergo slowdeamidation of the asparagine residue into isoaspartic acid.The resulting isoDGR peptides, like RGD peptides, bind to avintegrins. Our results exclude integrin involvement in the

binding of iNGR peptide and phage to cultured cells. It is alsounlikely that isoDGR formation affects the in vivo tumortargeting of iNGR because the deamidation process takesseveral hours (24), whereas the half-life of intravenouslyinjected peptides of the size of iNGR is only minutes (25).Thus, iNGR and iRGD bind to different primary receptorson cells.

Upon engagement of the iRGD peptide at the plasmamembrane of target cells, a proteolytic cleavage by a yet-to-be-identified enzyme(s) exposes the CendR sequence, whichsubsequently binds to NRP-1 (4). Our evidence indicates thatthe same mechanism operates with iNGR. First, phage dis-playing the predicted CendR product of iNGR, CRNGR (iNGRt)bound to NRP-1 and NRP-2, and did so with a higher affinitythan CRGDK fragment of iRGD. The reason for the differencemay be that a peptide with a C-terminal arginine binds moreefficiently toNRPs than a peptidewith a lysine C-terminus (26).Comparison of the tumor-homing efficacy of iRGD with anarginine or a lysine (CRGDK/RGPDC) showed that the lysine-containing form was more effective in vivo (4). It may be thatother effects of the lysine residue, such as stronger integrinbinding or higher susceptibility to protease cleavage, overcomethe effect of lower affinity for NRPs. Second, iNGR, both as asynthetic peptide and on phage was taken up by cells in anNRP-dependent manner. Third, we isolated the iNGRt CendRfragment from inside cells treated with the intact iNGR pep-tide, as has been previously done with iRGD (4). Fourth, theco-injection of iNGR phage with neutralizing anti-NRP-1antibody resulted in a reduced extravasation of iNGR. Theseresults show that iNGRt, the active form of iNGR, is generatedthrough proteolysis and that the tumor-penetrating proper-ties of iNGR are based on its ability to activate the CendRpathway.

The activation of iNGR into iNGRt appears to take placeonly in tumors because iNGR only accumulated in tumors.In contrast, the truncated iNGRt form, while showing pre-ferential homing to tumors, also accumulated in the lungsand heart. This homing pattern reflects the expression ofNRP-1, which is universal in the blood vessels but particu-larly high in tumor vessels (27). The reason for the selectiveactivation of the cryptic CendR motif in tumors is likely to bethat binding to the primary receptor is needed for the activ-ating proteolytic cleavage. Previous work from our labora-tory has shown that an iRGD variant that does not bind tointegrins, but contains a CendR motif, does not penetrateinto cultured cells, whereas iRGD does (4). The nature ofthe primary receptor does not seem to matter, as long as thereceptor is tumor specific. iRGD and iNGR bind to differentprimary receptors, but both become activated in cell cul-tures and in tumors. Moreover, we have recently shown thata previously identified tumor-homing peptide, CGNKRTRGC(LyP-1; ref. 28) also penetrates into tumors through theCendR/NRP mechanism (6). The primary receptor for thispeptide is p32/gC1qR/HABP1, a mitochondrial proteinexpressed at the cell surface in tumors (29). Thus, our resultsshow that at least 3 different primary receptors can initiatethe sequence of events that leads to the NRP-dependentactivation of the CendR pathway in tumors. Importantly, the

Figure 5. iNGR enhances efficacy of anticancer drugs withoutaffecting side effects. A, mice bearing orthotopic 4T1 tumors weretreated every other day with PBS or 3 mg/kg of doxorubicin (DOX)combined with 4 mmol/kg of CNGRC or iNGR peptide. Tumorgrowth was assessed every other day. B, body weight changes ofthe tumor mice from the treatment studies (A). Percentage of bodyweight shift is shown. �, P < 0.05; 2-way ANOVA; ��, P < 0.001;2-way ANOVA. Error bars, SE.

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difference in the primary receptors allows us to differentiallytarget tumors or tumor areas based on receptor expressionpatterns, providing multiple options to enhance tumortherapy with tumor-specific CendR peptides.The experiments with phage, fluorophore-labeled peptide,

and nanoparticles showed the ability of iNGR to take coupledpayloads into the extravascular tumor tissue. Our resultsfurther show that such enhanced delivery and tumor penetra-tion also applies to compounds co-administered with iNGR.Importantly, we showed this for doxorubicin, the antitumoractivity of which was increased by injecting the drug togetherwith iNGR.The co-administration strategy has significant advantages.

First, because chemical coupling is not needed, new chemicalentities are not created, providing a faster route for clinicaldevelopment. Second, unlike targeting of compounds chemi-cally coupled to a homing element, the co-administrationprocess is not strictly dependent on the number of availablereceptors, which seriously limits the amount of a drug that canbe delivered to a target (30).Taken together, our results show that iNGR possesses

the same targeting ability as CNGRC, supplemented withcell-internalizing and tumor-penetrating properties. Thistransformation suggests an important principle: a targetingpeptide can be ad hoc improved by the addition of a CendRmotif, which endows the peptide with tissue-penetratingproperties and allows enhanced delivery of co-administeredcompounds into a target tissue. Rational optimization oftargeting peptides in this manner may also have valuableapplications in other diseases.

Disclosure of Potential Conflicts of InterestL. Alberici, C. Bordignon, C. Traversari, and G.-P. Rizzardi are employees of

MolMed S.p.A. K.N. Sugahara, V.R. Kotamraju, T. Teesalu, and E. Ruoslahti areshareholders of CendR Therapeutics Inc, which has rights to some of thetechnology described in the article. No potential conflicts of interest weredisclosed by the other authors.

Authors' ContributionsConception and design: L. Alberici, K.N. Sugahara, L. Agemy, T. Teesalu,C. Traversari, G.-P. Rizzardi, E. RuoslahtiDevelopment of methodology: L. Roth, K.N. Sugahara, T. Teesalu, G.-P.RizzardiAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): L. Alberici, L. Roth, K.N. Sugahara, L. Agemy,T. TeesaluAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): L. Alberici, L. Roth, K.N. Sugahara, L. Agemy,T. Teesalu, E. RuoslahtiWriting, review, and/or revision of the manuscript: L. Alberici, K.N.Sugahara, T. Teesalu, G.-P. Rizzardi, E. RuoslahtiAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): K.N. Sugahara, E. RuoslahtiStudy supervision: K.N. Sugahara, T. Teesalu, C. Bordignon, G.-P. Rizzardi,E. RuoslahtiPeptide synthesis: V.R. Kotamraju

Grant SupportThis work was partially supported by MolMed S.p.A. (www.molmed.com),

grants W81XWH-09-1-0698 and W81XWH-08-1-0727 from the USAMRAA of theDoD (E. Ruoslahti), and by grant R01 CA 152327 from the National CancerInstitute of the NIH. E. Ruoslahti is supported in part by CA30199 the CancerCenter Support Grant from the National Cancer Institute of the NIH.

The costs of publication of this article were defrayed in part by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 30, 2012; revised September 19, 2012; accepted October 14, 2012;published OnlineFirst November 14, 2012.

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Alberici et al.





2013;73:804-812. Published OnlineFirst November 14, 2012.Cancer Res Luca Alberici, Lise Roth, Kazuki N. Sugahara, et al.

Design of a Tumor-Penetrating PeptideDe Novo

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