paclitaxel enhances therapeutic efï¬cacy of the f8-il2

Therapeutics, Targets, and Chemical Biology

Paclitaxel Enhances Therapeutic Efficacy of the F8-IL2Immunocytokine to EDA-Fibronectin–Positive MetastaticHuman Melanoma Xenografts

Michele Moschetta1, Francesca Pretto1, Alexander Berndt4, Kerstin Galler4, Petra Richter4, Andrea Bassi3,Paolo Oliva1, Edoardo Micotti2, Giovanni Valbusa5, Kathrin Schwager6, Manuela Kaspar6, Eveline Trachsel6,Hartwig Kosmehl8, Maria Rosa Bani1, Dario Neri7, and Raffaella Giavazzi1

AbstractThe selective delivery of bioactive agents to tumors reduces toxicity and enhances the efficacy of anticancer

therapies. In this study, we show that the antibody F8,which recognizes perivascular and stromal EDA-fibronectin(EDA-Fn), when conjugated to interleukin-2 (F8-IL2) can effectively inhibit the growth of EDA-Fn–expressingmelanomas in combination with paclitaxel. We obtained curative effects with paclitaxel administered before theimmunocytokine. Coadministration of paclitaxel increased the uptake of F8 in xenografted melanomas,enhancing tumor perfusion and permeability. Paclitaxel also boosted the recruitment of F8-IL2–induced naturalkiller (NK) cells to the tumor, suggesting a host response as part of the observed therapeutic benefit. In support ofthis likelihood, NK cell depletion impaired the antitumor effect of paclitaxel plus F8-IL2. Importantly, thiscombination reduced both the tumor burden and the number of pulmonarymetastatic nodules. The combinationdid not cause cumulative toxicity. Together, our findings offer a preclinical proof that by acting on the tumorstroma paclitaxel potentiates the antitumor activity elicited by a targeted delivery of IL2, thereby supporting theuse of immunochemotherapy in the treatment of metastatic melanoma. Cancer Res; 72(7); 1814–24.�2012 AACR.

IntroductionMelanoma is themost commonnonhematopoietic cancer in

young adults, and the incidence of the disease is increasing atan alarming pace worldwide (1). In a meta-analysis of 42 phaseII cooperative group trials, median survival in metastatic stageIV melanoma was only 6.2 months, with a mean 1-year overallsurvival (OS) rate of 25.5% regardless of treatment regimen (2).Despite the recent success of targeted therapies against theBRAF protein—led by vemurafenib (PLX 4032; ref. 3)—meta-static melanoma remains a solid malignancy that deservestherapeutic options.

In the arena of chemotherapy dacarbazine is considered thebenchmark treatment for advanced melanoma, despite aresponse rate of less than 13% to 20% (4). Paclitaxel is areasonable second-line therapy that has been evaluated innumerous trials as monotherapy (5, 6) or as part of a combi-nation with other therapies (7). In metastatic melanoma,single-agent paclitaxel has antitumor effects comparable withthose reported for dacarbazine and other single-agent che-motherapies, with a response rate between 0% to 33% (6).

Immunotherapy is a well-recognized therapeutic tool forpatients with advanced melanoma. Interleukin-2 (IL2) is a U.S.Food and Drug Administration (FDA)-approved agent for thetreatment ofmetastaticmelanoma, whereas IFN-a is a therapyoption for adjuvant treatment of patients with resected mel-anoma. A meta-analysis reviewed 18 studies of chemotherapycombined with IL2 and IFN-a: although some of these studiesreported durable tumor responses or increased progression-free survival (PFS), no regimen to date has improved OS (8, 9).Moreover, these chemoimmunotherapy combinations are gen-erally associated with considerable toxicity.

Very recently ipilimumab, a monoclonal antibody (mAb),which blocks cytotoxic T-lymphocyte–associated antigen 4(CTLA-4) to potentiate T-cell antitumor response, was shownto improve OS in patients with previously treated metastaticmelanoma (10). The approval of ipilimumab by FDA hasrenewed interest in immunotherapy as a legitimate therapeu-tic approach for melanoma (11).

The selective delivery of bioactive agents (i.e., cytotoxicdrugs, radionuclides, or immunostimulatory cytokines) to the

Authors' Affiliations: Departments of 1Oncology and 2Neuroscience,Mario Negri Institute for Pharmacological Research; 3Dipartimento diFisica, Politecnico di Milano, Milan, Italy; 4Institute of Pathology, UniversityHospital Jena, Jena, Germany; 5Centro Ricerche Bracco–Bracco ImagingSpa, Colleretto Giacosa, Italy; 6PhilochemAG; 7Institute of PharmaceuticalSciences, ETH Z€urich, Z€urich, Switzerland; and 8Institute of Pathology,Helios-Klinikum Erfurt, Erfurt, Germany

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

M. Moschetta and F. Pretto contributed equally to this work.

Current address for M. Moschetta: Department of Internal Medicine andClinical Oncology, University of Bari-Medical School, Bari, Italy.

Corresponding Author:Raffaella Giavazzi, Department of Oncology, MarioNegri Institute for Pharmacological Research, Milan, Italy. Phone: 39-02-3901-4732; Fax: 39-02-3901-4734; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-11-1919

�2012 American Association for Cancer Research.

CancerResearch

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on November 18, 2018. © 2012 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from

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tumor site is a promising avenue for the development of novelanticancer therapies. Vascular and tumor stroma–associatedmarkers are particularly attractive targets for antibody-baseddelivery of therapeutic agents in view of their selective andaccessible expression in solid tumors (12).Tumor progression is generally accompanied by angiogen-

esis together with the reorganization of the extracellularmatrix (ECM). In this regard, the de novo synthesis of oncofetalisoforms of fibronectin (Fn) has been extensively describedduring tissue remodeling (13). Fn variants are generated byalternative splicing of the ED-A, ED-B, and IIICS domains, aswell as by de novo glycosylation. Recently the Fn isoformcontaining EDA has been shown to be associated to vascularand stromal structures in a wide variety of cancer types.Furthermore, Natali and colleagues described an abundantexpression of EDA-Fn in primary melanomas and their metas-tases (14). A high-affinity human mAb directed to EDA-Fn, F8,has been shown to display tumor-targeting selectivity inbiodistribution experiments (15). Moreover we have recentlyreported that this antibody fused to human IL2 (F8-IL2)improves the therapeutic performance of sunitinib, an inhib-itor of angiogenesis, in a xenograft model of human kidneycancer (16).In this study, we show that human metastatic melanoma

lesions express high levels of perivascular and stromal EDA-Fn, which is detectable with the recombinant antibody F8,and that the immunocytokine F8-IL2 administrated in com-bination with paclitaxel significantly inhibits [60%–80%complete regressions (CR)] the growth of a human xeno-grafted melanoma expressing a high level of EDA-Fn in nudemice. Furthermore F8-IL2 added to paclitaxel significantlyreduced metastatic nodules to the lung of mice bearingmetastatic melanoma. In vivo optical and dynamic contrastenhanced-MRI (DCE-MRI) and immunohistochemical dataindicate that the ability of paclitaxel to potentiate theantitumor activity of F8-IL2 is associated with its capabilityto modify certain tumor vessel functionalities related tovascular perfusion and permeability and to promote hostimmune reaction. Our findings endorse the use of chemoim-munotherapy in the treatment of metastatic melanoma andsuggest that paclitaxel is endowed with properties affectingthe tumor stroma that deserve attention for the design ofnovel combination treatments.

Material and MethodsCell lines, drugs, and reagentsThe A375M is a metastatic variant derived from the A375

humanmelanoma (American Type Culture Collection; ref. 17);this cell line was characterized by short-tandem repeat pro-filing (AmpFlSTR Identifiler Plus PCR Amplification kit;Applied Biosystems) and compared with DNA fingerprintingdatabases. WM1552, WM115, and WM983A cell lines, derivedfrom patient melanomas, were kindly provided by Dr. Meen-hard Herlyn (Wistar Institute, Philadelphia, PA; ref. 18); theWM1552/5 is a tumorigenic variant derived fromWM1552 (19)that underwent DNA fingerprinting analysis (as above) toconfirm the identity with the parental line.

Stocks of cell lines were stored frozen in liquid nitrogen andkept in culture for no more than 8 weeks before injection. Cellculture conditions are detailed in Supplementary Methods.

Paclitaxel (Indena S.p.A.), dacarbazine (Sanofi Aventis),recombinant human IL2 (IL2, Proleukin; Novartis), immuno-cytokine F8-IL2 (16), the isotype control KSF-IL2 (16), (SIP)F8-Alexa Fluor 750 (15), and the anti-asialo GM1 antiserum (WakoChemical GmbH) were prepared and delivered as described inSupplementary Methods.

Tumor samplesSpecimens of melanomas were from patients at diagnosis;

ethical approval was obtained from the Local Research EthicsCommittee of the Friedrich Schiller University, Jena, Germany.Xenograft specimens were from human melanoma growingintradermal in nudemice (20). The spatial distribution and therelation of EDA-Fn to vascular structures were shown with adoublefluorescence labeling procedure for F8 reactive-EDA-Fnand CD31 (21), as described in Supplementary Methods.

Mice and xenograft tumor modelsSix- to 8-week-old female NCr-nu/nu mice (Harlan) were

used. Procedures involving animals and their care were con-ducted in conformity with institutional guidelines that complywith national and international laws and policies (22, 23).

Orthotopic melanoma modelWM1552/5 (2� 106 cells) was transplanted subcutaneously

in the flank of nude mice which were then randomized fortreatment (N ¼ 10–12 per group) when tumors reachedapproximately 100 to 150mg.Micewere treatedwith paclitaxel(20 mg/kg i.v.), dacarbazine (160 mg/kg i.p.), F8-IL2 (20mg/mouse i.v.), KSF-IL2 (20 mg/mouse i.v.), or unconjugatedrecombinant IL2 (6.6 mg/mouse i.v) as single agent or incombination regimens. For the depletion of natural killer (NK)cells, anti-asialo GM1 antiserum (200 mL/1:10 dilution i.v.) wasadministered along the therapy. Schedule of treatments aredetailed in the Results. Animal management and data collec-tion were carried out with the help of the Study Director 1.8software (Studylog System, Inc.). Results were plotted as themean tumor weight against days after tumor randomizationand the efficacy of treatment was expressed as tumor weightand tumor growth delay (T�C¼median time to reach 500mgof treated tumor � median time to reach 500 mg of controltumor; ref. 22). CR was defined by absence of tumor for at least3 months. Toxicity was monitored by recording body weightloss.

Metastatic melanoma modelA375M (10 � 105 cells) was injected intravenously in the

tail vein of nude mice which were then randomized fortreatment (N ¼ 10 per arm) 7 days later. Mice were treatedwith paclitaxel, F8-IL2 (same dose as above) as single agentor in combination regimens as detailed in Results. Mice werekilled and lung harvested after 6 weeks of treatment. Metas-tases were counted blinded by 2 independent investigatorsand expressed as number and size of tumor nodules in thelung (17). The procedures for subcutaneous tumor and

Paclitaxel with F8-IL2 Immunocytokine Inhibits Melanoma

www.aacrjournals.org Cancer Res; 72(7) April 1, 2012 1815




metastasis evaluation are detailed in SupplementaryMethods.

In vivo imagingUptake of (SIP)F8-Alexa Fluor 750 inmice bearingWM1552/

5 tumors was analyzed with the eXplore Optix imaging system.DCE-MRI was done after injection of the B22956/1 contrastagent with a BioSpec 70/30 AVIII system (24). Data analysis isin Supplementary Methods.

Histologic analysesAnalysis was carried out on WM1552/5 tumors from the

different treatment regimens harvested 24 hours after the endof the third cycle (N ¼ 4/5 tumors per group). For vesselperfusion analysis, mice were given Hoechst 33342 (40 mg/kgi.v; Sigma) 1 minute before sacrificing. Tumor sections wereCD31 stained for immunofluorescence microscopy (22) andimmunostained with the antibody against Asialo-GM1 for thedetection of tumor-infiltrating NK cells. Evaluation analysis isdescribed in Supplementary Methods.

ResultsEDA-Fn expression in metastatic melanomas andselection of the xenograft models

Tumor specimens obtained from patients with metastaticmelanoma were evaluated for EDA-Fn expression using thehuman recombinant SIP format antibody F8. Melanomas ingeneral showed an abundant deposition of F8 accessible EDA-Fn antigen in tumor vessels, aswell as in the tumor interstitiumor in the stroma. Figure 1A and B show representative lymphnode metastases of a malignant melanoma with a polygonalepitheloid/partially spindle shaped cell appearance and abun-dant stroma induction (Fig. 1A) and of a malignant melanomawith the typically medullary growth pattern (Fig. 1B). Bothare characterized by a strong vascular and stromal positi-vity for EDA-Fn. The expression of EDA-Fn was studied in apanel of human melanoma–derived xenografts, transplantedorthotopically in the derma of nude mice. Interestingly,the 4 human melanoma xenografts were characterized by ahighly diffuse EDA-Fn expression that fully replicated thatof the patient lesion (Fig. 1C–F). The WM1552/5 and theA375M metastatic melanoma were chosen to evaluate thetherapeutic activity of the F8-IL2 immunocytokine in preclin-ical models.

The antitumor efficacy of F8-IL2 immunocytokine isenhanced by the combination with paclitaxel in humanmelanoma xenografts

F8-IL2 alone and in combination regimens was tested onhuman tumor xenografts. Treatments were done for 5 cycleswith chemotherapy administered 24 hours before the immu-noconjugate (Treatment schedule in Fig. 2A).

Figures 2B and C show the therapeutic effects of F8-IL2in combination with paclitaxel or dacarbazine on WM1552/5melanoma growing subcutaneously in nude mice. F8-IL2 andIL2 administered as single agents did not significantly affecttumor growth. Paclitaxel treatment caused a marginal, though

significant, delay in growth compared with controls (T-C¼ 8);this delay was not improved by the addition of IL2 (T-C¼ 10).Only the addition of F8-IL2 to paclitaxel yielded a clear anti-melanoma activity and induced the complete eradication ofWM1552/5 tumors (Fig. 2B). Nine of 11 (82%)mice were tumorfree, as confirmed by histologic analysis at necropsy. Theinjection of F8 antibody plus, but not conjugated to, IL2 hadno effect on tumor growth (data not shown).

By contrast, in a similar trial carried out with dacarbazine incombination with F8-IL2 (Fig. 2C), no significant therapeuticbenefit was observed by combining the 2 treatments, despitethe ability of dacarbazine to cause a significant, albeit limited,growth delay as single agent (T-C ¼ 4 and T-C ¼ 6, fordacarbazine alone and in combination, respectively). Theadministration of IL2 conjugated to KSF that recognizes anunrelated antigen (KSF-IL2), in combination with paclitaxel,did not show advantage compared with paclitaxel alone(Fig. 2D).

Melanoma patient, case 1 Melanoma patient, case 2

WM1552/5 melanoma xenograft WM115 melanoma xenograft

A375M melanoma xenograft WM983A melanoma xenograft

BA

DC

FE

Figure 1. EDA-Fn expression in human melanoma. A and B, metastaticmelanomapatient lesions characterized by a strong vascular and stromalpositivity for EDA-Fn as shown by immunofluorescence double labeling.C to F, WM1552/5, WM115, A375M, and WM983A human melanomaxenografts are characterized by a highly diffuse EDA-Fn deposition thatfully replicated that of the patient lesion. Green fluorescence, EDA-Fn;red fluorescence, CD31. Bars, 200 mm.

Moschetta et al.

Cancer Res; 72(7) April 1, 2012 Cancer Research1816




Noteworthy was the finding that the therapeutic perfor-mance of the combination therapies was not associated withany significant loss of body weight, indicating that the com-bination therapy regimens were well tolerated.

Paclitaxel increases the uptake of F8 antibody inmelanoma xenografts

The influence of paclitaxel on the delivery of F8-IL2 wasinvestigated in vivo by optical imaging of (SIP)F8-Alexa Fluor750 tumor uptake in nude mice bearing subcutaneousWM1552/5 and pretreated with paclitaxel, dacarbazine orvehicle. Figure 3A shows representative images of vehicle-,dacarbazine-, and paclitaxel-treatedmice, the latter presentinga distinguishably higher fluorescence signal, which correspondto a stronger uptake of (SIP)F8-Alexa Fluor 750 in the tumor,compared with vehicle- or dacarbazine-treated mice. To eval-uate the uptake of (SIP)F8-Alexa Fluor 750 in tumors accordingto the different tumor weights (vehicle, mean ¼ 172 mg;paclitaxel, mean ¼ 91 mg; dacarbazine, mean ¼ 136 mg; N¼ 4 per group), the fluorescence signals were calculated as theratio between photon counts and tumor weights (Fig. 3B).Signals in the tumors of mice treated with paclitaxel wassignificantly higher compared with vehicle- or dacarbazine-treated animals, for both acquisitions done at 6 and 24 hoursafter (SIP)F8-Alexa Fluor 750 injection.

Paclitaxel increases tumor vessel perfusion andpermeability in melanoma xenografts

To elucidate whether the paclitaxel-induced increase of F8antibody uptake could be attributable to an altered tumorphysiology (25), perivascular Hoechst 33342 perfusion wasmeasured in tumors from animals treated with paclitaxel 24hours before (Fig. 3C). The area perfused by Hoechst 33342 wassignificantly higher in paclitaxel-treated tumors comparedwith controls (Fig. 3D). The injection of F8-IL2 or IL2 alonedid not modify the perfusion of Hoechst 33342 in tumors (datanot shown).

To obtain a more detailed analysis of tumor vessel func-tionality, paclitaxel- and vehicle-treated nude mice bearingWM1552/5 melanoma xenografts (N¼ 4 mice per group) wereanalyzed by DCE-MRI 24 hours after paclitaxel administration(Fig. 3E–G). To prevent perfusion and permeability from beinginfluenced by the size of lesions, paclitaxel- and vehicle-treatedtumors were weight-matched (mean ¼ 603 mg and 503 mg,respectively).

The kinetics of the distribution of the contrast agent(B22956/1) in the tumor core and rim were different. Tumorrim, a 1-mm thick band starting from the tumor boundary, isknown to be hypervascularized whereas the core is oftencharacterized by the presence of necrotic areas (Fig. 3E I).

After paclitaxel treatment a significant increase in thefractional plasma volume (fPV), an MRI-based measure oftumor perfusion, and transfer coefficient (kTrans), an MRI-derived quantity related to the extravasation of the contrastagent, was observed both in tumor rim and core, suggestingthat the treatment with paclitaxel was able to enhance theoverall delivery of the albumin-binding contrast agent to thepathologic tissue (Fig. 3E–G; ref. 26).

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Tumortransplantation Randomization Assessment of tumor response

Figure 2. Therapeutic activity of F8-IL2 immunocytokine in combinationwith chemotherapy in human melanoma xenografts. Nude mice bearingestablished (100–150 mg subcutaneous) WM1552/5 melanoma weretreated (every 4 days for 5 cycles) with paclitaxel (20 mg/kg i.v.),dacarbazine (160 mg/kg i.p.), F8-IL2 (20 mg/mouse i.v.), KSF-IL2(20 mg/mouse i.v.), or unconjugated recombinant IL2 (6.6 mg/mouse i.v.)as single therapy or in combination regimens (paclitaxel ! F8-IL2;paclitaxel ! KSF-IL2; paclitaxel ! IL2; dacarbazine ! F8-IL2).Chemotherapy was administered 24 hours before immunocytokine. A,protocol of the trials indicating cycles of treatment (arrows). B, C, and D,trial onWM1552/5melanoma treatedwith (B) paclitaxel, F8-IL2, or IL2; (C)dacarbazine and F8-IL2; and (D) paclitaxel and KSF-IL2 as single therapyor in combination regimens. Small triangles indicate cycles of treatment.N ¼ 10–12 mice per group. CR, complete regression.�, P < 0.05;��, P < 0.01; ��, P < 0.001 (2-way ANOVA test followed by Bonferronipost-test). DTIC, dacarbazine; PTX, paclitaxel.






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Figure 3. Paclitaxel induced functional modifications in tumor vessels and promoted tumor perfusion and F8 antibody uptake inWM1552/5melanomas. Nudemice bearingWM1552/5melanomaswere treatedwith 3 cycles of paclitaxel (20mg/kg, i.v.) or dacarbazine (160mg/kg i.p.) 24 hours before (A andB) receiving(SIP) F8-Alexa Fluor 750 (60 mg i.v) or (C, D) being perfused with Hoechst 33342 (40mg/kg i.v, oneminute before sacrificing) or (E, F, and G) analyzed by DCE-MRI. A, images representative of F8-Alexa Fluor 750 selectively accumulated in WM1552/5 subcutaneous tumor with a distinguishably larger fluorescencesignal in paclitaxel-treated mice compared with dacarbazine-treated mice. The color bar shows the fluorescence intensity (photon counts) in the acquisitionregion. B, fluorescence signal was expressed as the ratio between photon counts and tumor volumemeasured at 6 and 24hours after (SIP) F8-Alexa Fluor 750administration (a.u., arbitrary units). Values are mean� SD; N¼ 4. �, P < 0.05; ��, P < 0.01 compared with vehicle (2-way ANOVA test followed by Bonferronipost-test). C, representative pictures of Hoechst 33342 perfusion showing an increased perivascular/interstitial accumulation of the dye in the WM1552/5xenograft section from paclitaxel-treated animals compared with time-matched vehicle-treated mice. D, graphical presentation of the semiquantitativeassessment of the perivascular/interstitial Hoechst 33342 staining. Values aremean�SD;N¼5. ��,P <0.01 (Mann–WhitneyU test). E, I, representative imageof tumor core and rim regions of interest. E, II and III, parametric images of kTrans superimposed over anatomical MRI images for a vehicle and a paclitaxel-treated tumor. F andG, box plots of DCE-MRI pharmacokinetic parameters (fPV and kTrans) in tumor rim (F) and core (G) 24 hours after paclitaxel.N¼ 4; opencircles represent outlier values.�, P < 0.05; ��, P < 0.01 compared with vehicle (Mann–Whitney U test). DTIC, dacarbazine; PTX, paclitaxel.

Moschetta et al.





Altogether, these data suggested that paclitaxel inducedtransient functional modifications in tumors vessels, whichin turn could promote F8-IL2 delivery and tumor response.

The therapeutic efficacy of the F8-IL2 immunocytokinecombined with paclitaxel is sequence dependentTo evaluate the impact of the findings above on drug

scheduling and tumor response, additional trials comparingdifferent sequences of drug administration were carried out(Fig. 4). The time interval between treatments was prolongedto 7 days to allow clearance of circulating F8-IL2. In this settingpaclitaxel as single agent only moderately affected the growthof WM1552/5 human melanoma xenografts (T-C ¼ 7). In thecombination arm, the weekly schedule of F8-IL2 given 24 hoursafter paclitaxel (paclitaxel ! F8-IL2) significantly inhibitedWM1552/5 melanoma growth, with 5 of 11 (45.5%) CR,thus confirming the findings above (Fig. 4A). In the same study,

F8-IL2 administered 4 hours after paclitaxel (paclitaxel þF8-IL2) showed a comparable therapeutic efficacy with 6 of11 (54.5%) CR. Curedmice were still tumor-free after 4months,when the experiment was terminated and mice necropsied.Noncured tumors, after a significant growth delay (T-C¼ 29; 6tumors for paclitaxel ! F8-IL2 and T-C ¼ 30; 5 tumors forpaclitaxelþ F8-IL2), resumed their growth at the same rate ascontrol-untreated tumors. By contrast, the efficacy of thecombination was reduced with the inverted sequence of drugadministration (F8-IL2 ! paclitaxel; paclitaxel administered48 hours after F8-IL2), but not significantly different frompaclitaxel alone (Fig. 4B). With no mice being tumor-free atthe end of the study, no CRs were observed in this trial.

Effect of F8-IL2 and paclitaxel on host cell infiltrationThe contribution of host immune cells to the therapeutic

effects of F8-IL2 combined with paclitaxel was analyzed inWM1552/5 melanoma sections harvested from mice 24 hoursafter the third cycle of treatments. F8-IL2 administered afterpaclitaxel led to amassive infiltration of NK cells inWM1552/5tumors that was significantly higher than in lesions treatedwith paclitaxel or F8-IL2 as single agents (Fig. 5A and B),whereas the inverted sequence with F8-IL2 administered afterpaclitaxel did not enhance NK cell infiltration (Fig. 5C and D).Accordingly, IL2 had no effect on NK cell infiltration, and F8-IL2 alone caused only a marginal recruitment. Notably, asignificant increase of NK cells was seen in the paclitaxel-onlytreated tumors (Fig. 5A–D), suggesting that paclitaxel isendowed with immunomodulating properties (27). The hostinfiltration of controls at randomization (day 0, mean tumorweight ¼ 120 mg) and on the day of testing (mean tumorweight ¼ 500 mg) was not different.

To examine the implication of NK cells on the growthof WM1552/5, nude mice undergoing therapy with pacli-taxel ! F8-IL2 were depleted of NK cells with anti-asialoGM1. Figure 5E shows that in mice receiving anti-asialoGM1, paclitaxel ! F8-IL2 was significantly less efficaciousin inhibiting the growth of WM1552/5 xenografts andnot significantly different from paclitaxel alone. Anti-asialoGM1 given to vehicle-treated tumor did not influencetumor growth. These findings point to the contributionof the host response to the therapeutic benefit of thecombination.

Paclitaxel combined with F8-IL2 reduced distantmetastasis by melanoma

The therapeutic efficacy of F8-IL2 immunocytokine in com-bination with paclitaxel was assessed on lung tumor nodulesformed by the A375M melanoma. Treatment started 7 daysafter tumor injection, when tumor foci were established in thelung of nude mice (17). According to the findings abovepaclitaxel was given 24 hours before F8-IL2 and therapyrepeated weekly for 6 cycles. As shown in the representativelungs of Fig. 6A, F8-IL2 did not affect metastasis, whereaspaclitaxel-based chemotherapy inhibited the growth of tumornodules (tumor burden, Fig. 6C), but not the overall number ofcolonies in the lung (colony number, Fig. 6B). Only the com-bination of paclitaxel with F8-IL2 significantly reduced both

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Figure 4. The efficacy of the combination F8-IL2 plus paclitaxel dependson the sequence of treatment administration. Nude mice bearingsubcutaneously established (150 mg) WM1552/5 melanoma weretreated (every 7 days for 4 cycles) with paclitaxel (20 mg/kg i.v.) or F8-IL2(20 mg/mouse i.v.), as single therapy or in combination regimens,according to the following schedules: (A) paclitaxel administered 24 or 4hours before F8-IL2 (paclitaxel ! F8-IL2 and paclitaxel þ F8-IL2); (B)paclitaxel administered 48 hours after F8-IL2 (F8-IL2! paclitaxel). Smalltriangles indicate cycles of treatment. N ¼ 8–11 mice per group. CR,complete response. �,P < 0.05; ��,P < 0.01; ��,P < 0.001 (2-way ANOVAtest followed by Bonferroni post-test).






number and tumor burden of lung colonies (Fig. 6B and C andSupplementary Table S1).

DiscussionOur study shows in mouse xenograft models of human

melanoma that paclitaxel has a synergistic antitumor effectwith the immunocytokine F8-IL2 by favoring its tumor uptakeand potentiating its antitumor efficacy. This treatment modal-ity induced CR of the WM1552/5 melanoma transplantedsubcutis in nude mice and inhibited metastasis to the lungfrom the A375M.

The therapeutic performance of the F8-IL2-paclitaxel seemsto be contingent on the pattern of positivity for EDA-Fn: inresponsive melanoma, this Fn isoform was highly expressed invessel walls and in the tumor interstitial space (Fig. 1). Atvariance the same combination was moderately active in amodel of ovarian cancer in which EDA-Fn expression wasrestricted to the perivascular space only (SupplementaryResults and Supplementary Fig. S1). Noteworthy, our findingsare in agreement with the results of Natali and colleagues,showing EDA-Fn expression associated with melanomametastatic lesions (14). Altogether these findings suggest

that vascular targeting based therapy should be adapted tothe individual features of patient's tumor; in the case ofmelanoma, this tumor type is putatively a fitting candidatefor immunoconjugated based therapy using the F8 antibody asa vehicle.

Our data also show that the therapeutic efficacy of F8-IL2 issignificantly improved by combining certain chemotherapeu-tic agents—here we show paclitaxel—but not others, such asdacarbazine. The increased uptake of fluorescent F8 antibodyin the tumors of mice pretreated with paclitaxel, but not withdacarbazine, supports its use in this combination setting.Because Hoechst 33342 staining used as an indicator of vas-cular perfusion (28) was increased in the tumors of micetreated with paclitaxel, we hypothesized an increase of tumorperfusion. Surprisingly, although IL2 had previously beenshown to increase vascular permeability in patient healthytissue and in tumor-bearing mice (29, 30), a similar effect wasnot observed in our studies when the fusion protein F8-IL2 wasused as single agent. The resistance of tumor tissue to IL2-induced increase vascular permeability could be expression ofthe angiogenic phenotype of tumor endothelial cells (moreresistant to IL2 direct toxic effect) or to the different cytokinemilieu of tumor tissue.

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Figure 5. Tumor-infiltrating host cells contribute to the antitumor effect of F8-IL2. A to D, immunohistochemistry analysis. Twenty-four hours after the thirdcycle of treatment (as inFig. 4),WM1552/5 xenograft tumorswere harvestedandsections immunostained.AandB, protocolwithpaclitaxel!F8-IL2;CandD,protocol with F8-IL2! paclitaxel. A and C, representative images of tumor section stained for Asialo-GM1 antibody (NK cells) from the different treatmentgroups. B and D, percentage of stained area to total image area was assessed by computer-aided image analysis; data are mean� SD of 5 randomly chosenpictures from at least 5 different tumors per group. �, P < 0.05; ��, P < 0.01; ��, P < 0.001 (Kruskal–Wallis test). E, effect of NK depletion on tumorgrowth. Anti-asialoGM1wasgiven tomicebearingWM1552/5 xenograft (tumorweight¼120mg) starting 4daysbefore treatment began (paclitaxel!F8-IL2)and every 4 days thereafter until the end of the third cycle of treatment. Data (mean � SD of 8 tumors) represent tumor weight at 2 weeks posttherapy.�, P < 0.05; ��, P < 0.01; ��, P < 0.001 (one-way ANOVA test followed by Bonferroni post-test).

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Subsequent DCE-MRI studies on mice pretreated with pac-litaxel showed a significant increase in tumor perfusion (fPV)and in vessel permeability (kTrans), suggesting that thesemodifications improve distribution of the immunocytokine.Of further note was our use as contrast agent of gadocoletic

acid trisodium salt (B22956/1), which belongs to the class ofalbumin binder agents. Given that the fractional binding of thegadocoletate ion to animal and human serum albumin isestimated at around 90% (31), we can conclude that in ourtumormodel paclitaxel is ultimately able to induce an increasein B22956/1-labeled albumin extravasations (represented bythe increase of kTrans values) partially through its ability toincrease fPV in the leaky tumor vessels. Themolecular weightsof the F8 antibody in SIP format (80 kDa; imaging study) and inscFv-IL2 format (86 kDa for the noncovalent homodimer usedfor the therapy study) are comparable with the one of albumin.It is reasonable to conclude that one of the therapeuticallyrelevant actions of paclitaxel consist inmediating an increasedvascular permeability at the tumor site. The increased vascularpermeability could be attributable to the effect of paclitaxel ontumor angiogenesis (32, 33). However at the dose/scheduleused in this study we did not find significant changes in vesselnumber, area, and diameter (Supplementary Fig. S2), soincreased perfusion/permeability is presumably not caused

by a direct effect on tumor vascularization. Instead our find-ings are in line with results from other Authors. Jain andcolleagues showed that paclitaxel is able to reduce tumorinterstitial fluid pressure (IFP) and decompress tumor bloodvessels. The observation that these effects are no more evidentin taxane-resistant tumors supports the hypothesis that thesolid stress generated by neoplastic cell proliferation deter-mines the increase in tumor vascular resistance (through thecompression of tumor vessels) and in IFP (34). Thus, a reduc-tion in tumor cell density through the use of a conventionalcytotoxic drug ultimately decompresses blood vessels and,hence, reducesmicrovascular resistance and IFP and increasestumor perfusion (34). However, the theory of solid stress intumors only partially explains the biologic mechanisms bywhich taxanes induce changes in vessel parameters. Indeed,another study showed that paclitaxel and docetaxel loweredIFP and significantly increased albumin extravasation regard-less of their cytotoxic activity, suggesting that these effectscould be taxane specific and related to pharmacodynamics ofthese drugs (35).

Elevated IFP is the major physiologic barrier to the deliveryof macromolecules. It reduces the minimal physiologic extrav-asation of albumin that normally accounts for interstitialoncotic pressure (36) and could explain the inadequate

Figure 6. F8-IL2 in combinationwithpaclitaxel inhibits metastasis in thelung. Nudemice injectedwith A375Mhuman melanoma (10 � 105 cells i.v)were treated 7 days later withpaclitaxel (20mg/kg i.v.) or F8-IL2 (20mg/mouse i.v.), as single therapy or incombination regimens (paclitaxel !F8-IL2) for 6 weekly cycles.Chemotherapy was administered 24hours before immunocytokine. Oneweek after treatment mice weresacrificed, lungs harvested inBouin's fixative, and tumor lungcolonies evaluated. A, representativeimages of lungs from each treatmentgroup. B and C, scatter plots ofcolony number (B) and volume (C) foreach lung. Horizontal bars, median.N ¼ 10 mice per group. �, P < 0.05;��, P < 0.01; ��, P < 0.001 (Kruskal-Wallis test). Results are from 2independent experiments.

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delivery and therapeutic efficacy of F8-IL2 when administeredas monotherapy. With the coadministration of paclitaxel, theantibody is likely able to reach the tumor at the right plasmaconcentration, to extravasate and accumulate in the perivas-cular and interstitial space of the tumor.

The translational potential of these findings is substantiatedby clinical studies. In breast cancer patients treated withneoadjuvant chemotherapy, paclitaxel decreased IFP andincreased oxygenation, whereas doxorubicin did not produceany significant change (25); this finding suggests that at leastthese tumors would be better treated first with paclitaxel toreduce IFP and increase pO2 to improve the delivery ofsubsequent therapy, in particular of large molecules such asantibodies. Further studies are needed to clarify the mecha-nism of this sequencing.

These findings, suggesting that tumors would be bettertreated with paclitaxel first to improve subsequent drug deliv-ery, have prompted us to evaluate the sequencing of paclitaxeland F8-IL2 to maximize tumor response. We found thatpaclitaxel administered 4 or 24 hours before F8-IL2 was moreefficacious than F8-IL2 first followed by paclitaxel. In thelast sequence paclitaxel was administered 48 hours after F8-IL2, a time sufficient for the clearance of the antibody fromcirculation. Here we used a recombinant antibody in scFvformat to develop an immunocytokine with adequate tumortargeting and rapid clearance (16, 37). Further investigations,however, will be necessary to ascertain whether paclitaxel canalso increase the tumor uptake of conventional IgG-basedtherapeutics.

In previous studies with IL2-based immunocytokines, theantitumor activity was traced principally to NK cells (16, 38).Here we found that F8-IL2 combined with paclitaxel induced amassive infiltration of NK cells in WM1552/5 tumors that wassignificantly greater than what occurred in xenografts treatedwith either of the 2 agents singly. We suggest that paclitaxel-caused increased permeability leads to the accumulation of F8-IL2 in the interstitium of the tumor (where abundant EDA-Fncan be found), resulting in a local activation and mitogenicactivity for NK cells. The hypothesis that NK cells mediate thetherapeutic activity of the immunocytokinewith paclitaxel wasreinforced by the observation that NK cell depletion in tumor-bearing mice abolishes the therapeutic gain associated to thecombined use of F8-IL2 with paclitaxel. Furthermore, anincrease of F4-80þmacrophages in tumors frommice receivingF8-IL2/paclitaxel (Supplementary Fig. S3) was also observed,thus calling for a more complex implication of the host in thetherapeutic activity of the immunocytokine.

Notably, a significant increase in Asialo-GM1þ NK cells wasalso observed in tumors treated with paclitaxel alone, suggest-ing that the taxane possesses immunomodulating properties.These findings speak for a role of paclitaxel in boosting thisresponse to F8-IL2. Indeed, there is an emerging body of dataconfirming that paclitaxel, like other related taxanes, displaysimmunomodulatory properties that endorse its therapeuticapplication beyond tumor chemotherapy (27, 39).

We are aware of the possible limits of studies done inimmunodeficient nude mice, in which most of the immuno-modulating effects were underestimated and restricted to

innate immunity (nude mice cannot generate mature T lym-phocytes; ref. 40). Indeed, in some tumor models, T cells havealso been shown to contribute to the action of IL2-basedimmunocytokines (41). Nevertheless, in a number of cancermodels based on the grafting of murine tumors in syngeneicmouse strains, the therapeutic activity of IL2-based immuno-cytokines was comparable when immunocompetent mice orimmunocompromised mice (which still had NK cells) wereused (38).

Attempts to treat melanoma patients with high dose IL2have been conducted for more than 2 decades (42). However,the modest benefits achieved by treatment regimens includingthis cytokine must be weighed against the serious side effects.In our study inmelanoma-bearingmice the targeted delivery ofIL2 to the tumor allowed repeated and well-tolerated admin-istrations of F8-IL2; more importantly, the administration incombination with paclitaxel did not seem to cause any cumu-lative toxicity. Paralleling our findings 2 different IL2-basedimmunocytokines (L19-IL2 and F16-IL2; refs. 37, 38) are cur-rently being investigated in phase II clinical trials for differentindications and in combinations with cytotoxic agents (43). Alarge variation in response to IL2-based immunocytokinetreatment, in combination with dacarbazine, has beenobserved in a recent phase IIa trial in patients with metastaticmelanoma, ranging from patients who did not respond totreatment to a set of patients (29%) who enjoyed a RECIST-confirmed partial or complete response (43). It is still unclearwhether these large interpatient differences are due to avariability in the uptake of the immunocytokine at site ofdisease, or rather to the immunologic features of individualpatients (e.g., different tumor-associated antigens presented bythe tumor, different cytokine environment, or different activityof T and NK cells).

In conclusion, these preclinical results point to a signif-icant benefit resulting from the combination of the immu-nocytokine F8-IL2 with paclitaxel for the treatment ofmetastatic melanoma, a tumor that historically has beenresistant to chemotherapy. Paclitaxel, when administeredfirst, seems to enhance the antibody-targeted delivery of IL2to tumors that selectively express EDA-Fn, thereby improv-ing the therapeutic index of the cytokine. We found thatpaclitaxel alone was able to affect the growth of metastaticlesions, but only its combination with F8-IL2 significantlyreduced tumor nodules in the lung of nude mice. Should ourpresent findings be confirmed by further investigations, theEDA-Fn expression pattern in lesions and the scheduling ofdrug administration will prove to be critical factors to takeinto account with this new biopharmaceutical agent. As thecontrol of visceral metastases remains a main challenge inthe treatment of melanoma patients (44), the therapeuticmodalities and results reported herein should have animpact on the design of immunocytokine-based clinicaltrials in patients with melanoma and other malignancies,in combination with chemotherapeutic agents.

Disclosure of Potential Conflicts of InterestD. Neri is cofounder and shareholder of Philochem, as well as owner of the F8

antibody. The other authors declared no potential conflicts of interest.

Moschetta et al.





Authors' ContributionsConception and design: M. Moschetta, F. Pretto, H. Kosmehl, M.R. Bani, D.Neri, and R. Giavazzi.Writing, review, and/or revision of themanuscript:M.Moschetta, F. Pretto,M.R. Bani, D. Neri, and R. Giavazzi.Acquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): F. Pretto, A. Berndt, K. Galler, P. Richter, A. Bassi, P.Oliva, and E. Micotti.Analysis and interpretation of data (e.g., statistical analysis, biostatistics,andcomputational analysis):F. Pretto, A. Berndt, K. Galler, P. Richter, A. Bassi,G. Valbusa, D. Neri, and R. Giavazzi.Development of methodology: P. Oliva and E. Micotti.Administrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): K. Schwager, M. Kaspar, and E.Trachsel.Production of F8-IL2 and KSF-IL2: K. Schwager.Study supervision: M.R. Bani and R. Giavazzi.Development of reagents for the execution of the therapy study: D. Neri.

AcknowledgmentsThe authors thank Antonietta Silini and Valentina Scarlato for technical

assistance.

Grant SupportThe research work was supported by the 7th EU Framework Program

(ADAMANT HEALTH-F2-2008-201342 to R. Giavazzi, D. Neri, and E. Trachsel),the Italian Association for Cancer Research (R. Giavazzi) and FondazioneCARIPLO (no. 2008-2264 to R. Giavazzi).

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicate thisfact.

Received June 9, 2011; revised January 26, 2012; accepted February 7, 2012;published OnlineFirst March 5, 2012.

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2012;72:1814-1824. Published OnlineFirst March 5, 2012.Cancer Res Michele Moschetta, Francesca Pretto, Alexander Berndt, et al. Melanoma Xenografts

Positive Metastatic Human−Immunocytokine to EDA-Fibronectin Paclitaxel Enhances Therapeutic Efficacy of the F8-IL2

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