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CANCER

Antibody-Based Delivery of Inteleukin-2to Neovasculature Has Potent ActivityAgainst Acute Myeloid LeukemiaKatrin L. Gutbrodt,1* Christoph Schliemann,2* Leonardo Giovannoni,3 Katharina Frey,1

Thomas Pabst,4 Wolfram Klapper,5 Wolfgang E. Berdel,2† Dario Neri1†AQ1

Acute myeloid leukemia (AML) is a rapidly progressing disease that is accompanied by a strong increase in micro-vessel density in the bone marrow. This observation prompted us to stain biopsies of AML and acute lymphoidleukemia (ALL) patients with the clinical-stage human monoclonal antibodies F8, L19, and F16 directed againstmarkers of tumor angiogenesis. The analysis revealed that the F8 and F16 antibodies strongly stained 70% ofAML and 75% of ALL bone marrow specimens, whereas chloroma biopsies were stained with all three antibodies.Therapy experiments performed in immunocompromised mice bearing human NB4 leukemia with the immuno-cytokine F8-IL2 [consisting of the F8 antibody fused to human interleukin-2 (IL-2)] mediated a strong inhibitionof AML progression. This effect was potentiated by the addition of cytarabine, promoting complete responses in40% of treated animals. Experiments performed in immunocompetent mice bearing C1498 murine leukemia re-vealed long-lasting complete tumor eradication in all treated mice. The therapeutic effect of F8-IL2 was mediatedby both natural killer cells and CD8+ T cells, whereas CD4+ T cells appeared to be dispensable, as determined inimmunodepletion experiments. The treatment of an AML patient with disseminated extramedullary AML manifes-tations with F16-IL2 (consisting of the F16 antibody fused to human IL-2, currently being tested in phase 2 clinicaltrials in patients with solid tumors) and low-dose cytarabine showed significant reduction of AML lesions andunderlines the translational potential of vascular tumor–targeting antibody-cytokine fusions for the treatment ofpatients with leukemia.

INTRODUCTION

The formation of new blood vessels (angiogenesis) is a rare process inthe healthy adult but represents an absolute requirement for the via-bility and growth of solid tumors (1, 2). Because this neovascular-ization is mediated by angiogenic molecules released by tumor cellsor by host cells (such as macrophages and lymphocytes), there is in-tense research activity aiming at the inhibition of tumor progressionby means of a molecular blockade of proangiogenic factors (3, 4).

In addition to the development of inhibitors of angiogenesis, asecond therapeutic strategy [“vascular targeting” (5, 6)] has been de-veloped to take advantage of tumor blood vessels for the selectivepharmacodelivery of potent therapeutic payloads (such as drugs, cy-tokines, radionuclides, photosensitizers, and toxins) to disease sites(7–12). Vascular tumor targeting relies on monoclonal antibodies, whichspecifically recognize markers on newly formed blood vessels. Markersof angiogenesis have historically been discovered by observing a selec-tive staining of newly formed blood vessels in large immunohisto-chemical screening campaigns (13–15). More recently, systematictranscriptomic (16) and proteomic (17–19) strategies have been devel-oped for the identification and validation of targets expressed at neo-vascular sites.

Our group has extensively studied the in vivo performance ofvascular-targeting antibodies in solid tumors in mouse models ofcancer (20–23) and in patients (24–26). Although we and others ini-tially focused on vascular-targeting approaches in solid tumors, we re-cently discovered that certain markers of angiogenesis are selectivelyand abundantly expressed in most lymphoma types and can be effi-ciently targeted in vivo using “armed” antibodies (25, 27–29).

Splice isoforms of fibronectin and of tenascin-C represent some ofthe best-characterized markers of angiogenesis (6, 30). Specifically, thealternatively spliced extradomains EDA and EDB of fibronectin, aswell as the extradomain A1 of tenascin-C, are virtually undetectablein normal adult tissues but are strongly expressed at sites of physio-logical angiogenesis and tumor angiogenesis (31). The human mono-clonal antibodies F8, L19, and F16 specifically recognize the EDA andEDB domains of fibronectin and the A1 domain of tenascin-C, respec-tively, and have been shown to selectively target tumors in mouse mod-els of cancer and in patients (24, 32–35). Although these antibodiesrecognize blood vessels of different cancer types (31, 36, 37), the ex-pression of other markers of angiogenesis appears to be more restrictedto hematological malignancies [for example, Bst-2 expression in lym-phomas (28)].

In 2000, Padró et al. reported that a high degree of neovascular-ization can be observed in the bone marrow of patients with AML.The authors found that microvessel density was strongly increasedin the bone marrow of AML patients compared to normal bone mar-row. Patients enjoying a complete remission (CR) after induction ther-apy exhibited a reduction of microvessel density to values comparableto the ones of control subjects, whereas higher vessel counts could bedetected in patients with residual disease (38).

1Department of Chemistry and Applied Biosciences, ETH Zürich, Wolfgang-Pauli-Strasse10, CH-8093 Zurich, Switzerland. 2Department of Medicine A, Hematology and Oncology,University Hospital Muenster, Muenster, GermanyAQ2 . 3Philogen SpA, Siena, Italy. 4Depart-ment of Medical Oncology, University Hospital and University of Berne, Berne, Switzer-land. 5Department of Pathology, Hematopathology Section and Lymph Node Registry,University of Kiel, Kiel, Germany.*These authors contributed equally to this work.†Corresponding author. E-mail: [email protected] (W.E.B.); [email protected](D.N.)

R E S EARCH ART I C L E

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Here, we analyzed the expression of the EDAand EDB domains of fibronectin and of A1 do-main of tenascin-C, which are undetectable inhealthy bone marrow (39), in freshly frozen bonemarrow and chloroma (extramedullary AML tu-mor) biopsies of AML and acute lymphoid leu-kemia (ALL) patients, showing significant stainingwith the F8 and F16 antibodies. The immuno-cytokine F8-IL2, consisting of the F8 antibodyfused to human interleukin-2 (IL-2) (33, 40), se-lectively localized to subcutaneously grafted AMLtumors and mediated substantial tumor growthretardation in mice. The combination of F8-IL2with cytarabine led to long-lasting tumor erad-ication in 40% of treated mice in an immuno-compromised mouse model, and 100% of treatedmice in an immunocompetent setting, in a pro-cess mediated by CD8+ T cells and natural killer(NK) cells. The findings promise to be of clinicalsignificance, because an AML patient, who hadpreviously relapsed from multiple lines of ther-apy and was then treated with the F16-IL2 im-munocytokine and low-dose cytarabine, exhibiteda rapid disappearance of 18F-fluorodeoxyglucosepositron emission tomography (18-FDG-PET)uptake in AML lesions (multiple chloromas).

RESULTS

Immunochemical analysis of bonemarrow biopsiesTwenty-one bonemarrow biopsies (17 AML and4 ALL patients) and 2 chloroma (AML) specimenswere freshly frozen and analyzed using the clinical-stage F8, L19, and F16 antibodies (patient charac-teristics are described in tables S1 and S2). The useof freshly frozen material was necessary becausethese antibodies do not work in paraffin (36, 41),thus preventing the use of larger collections offormalin-fixed, paraffin-embedded specimens.

F1 Figure 1 shows representative findings of animmunofluorescence and immunohistochemicalanalysis performed with a set of 13 AML speci-mens and 4 ALL specimens, which were col-lected in Muenster (additional staining resultsand patient characteristics can be found in fig.S1 and table S1). In addition to the F8, L19, andF16 antibodies, the KSF antibody [specific to henegg lysozyme (42)] was used as the negative con-trol. Blood vessels were costained using an anti-body specific to von Willebrand factor (vWF).In general, F8 and F16 exhibited the strongestimmunochemical staining, typically associatedwith vascular structures. Quantification of the im-munofluorescence data, based on the percentageof area stained per microscope field, revealedthat a considerable fraction of the specimens

Fig. 1. Splice isoforms of oncofetal fibronectin (EDA and EDB) and tenascin-C are expressedin bone marrow biopsies of acute leukemia patients. Sections of bone marrow biopsies of AMLand ALL patients were analyzed by immunofluorescence, as well as immunohistochemistry (IHC)using the clinical-stage F8, L19, and F16 antibodies (which recognize splice isoforms of oncofetalfibronectin and tenascin-C) (red) and the KSF antibody (which reacts with hen egg lysozyme andwas used as negative control) (red). In immunofluorescence procedures, blood vessels were addi-tionally costained using an antibody specific to vWF (green). (A and B) Representative stainings of(A) AML specimens and (B) ALL specimens. Scale bars, 100 mm. In general, F8 and F16 exhibited thestrongest immunochemical staining. The immunofluorescence stainings of leukemia patient biop-sies were quantified by means of the percentage area of staining for each antibody. (C) In AMLpatient biopsies, 9 of 13 specimens were substantially stained with F8, 9 of 13 with F16, and 5 of 13with L19. (D) ALL samples displayed strong staining in three of four cases for F8, three of four forF16, and two of four for L19, respectively.





analyzed (9 of 13 for F8, 9 of 13 for F16, and 5 of 13 for L19 in AML;3 of 4 for F8, 3 of 4 for F16, and 2 of 4 for L19 in ALL) displayed asubstantially stronger vascular staining with the tumor-targeting anti-bodies than the negative control antibody (Fig. 1, C and D).

Similar staining patterns were observed in an additional smaller setof four AML bone marrow biopsies, which were obtained from Berneor Kiel and analyzed by immunohistochemistry. In this set, three offour specimens were stained with F8, four of four with F16, and zeroof four with L19 compared to the negative control (fig. S2A).

Two specimens of chloromas (extramedullary tumors of AML)were also analyzed and showed clear vascular staining with F8, F16,and L19 (F2 Fig. 2 and fig. S2, B and C).

Immunochemical analysis of mouse models of leukemiaand biodistribution studiesThe L19 and F8 antibodies exhibit identical binding affinity towardthe cognate human and murine antigen (34, 35), whereas F16 doesnot cross-react with murine A1 domain of tenascin-C (32). For thisreason, we used the L19 and F8 antibodies to stain mouse leukemiasamples in a search for a suitable model in which to perform biodis-tribution and therapy studies.

In a first step, we subcutaneously implanted human AML cell linesNB4, HL60, and THP1 in nude mice. Immunofluorescence analysis ofthe resulting tumors showed an intense staining with the F8 antibodyin the NB4 and HL60 model. Staining intensities with L19, or in theTHP1 model, were weaker (F3 Fig. 3A). Subsequently, we established anorthotopic model of HL60 leukemia by intravenous injection of tumorcells in severe combined immunodeficient (SCID) mice, followed byanalysis of blasts in blood and bone marrow specimens. Unfortu-nately, unlike in the situation with human specimens, the bone mar-row of mice bearing HL60 leukemia did not exhibit any detectable F8staining, whereas leukemic cells could easily be detected using anti-human CD44 antibodies (fig. S3). For this reason, we continued ourinvestigations with the subcutaneous NB4, HL60, and THP1 AMLmodels in nude mice, which mimic the disease settings of chloromas.

Figure 3 (B to D) shows quantitative biodistribution results ob-tained by intravenous injection of radioiodinated preparations of theimmunocytokine F8-IL2 (40) (black bars) or of the anti-lysozyme KSF-IL2 fusion protein, used as negative control of identical molecularformat (white bars). A preferential accumulation in the subcutaneousneoplastic lesions was observed only for NB4 tumors, with 5.3% in-jected dose per gram of tumor 24 hours after injection and a tumor-to-blood ratio of 18.8. For this reason, the NB4 model was selected forsubsequent therapy experiments.

Therapy experiments in miceNude mice bearing subcutaneously grafted NB4 tumors (~50 mm3)were treated with intravenous injections of saline, F8-IL2, or KSF-IL2 (used as negative control immunocytokine) on days 7, 10, and13 (fig. S4, A and B). Substantial tumor growth retardation was ob-served up to day 23 in the case of F8-IL2 (but not KSF-IL2).

In a second experiment, immunocytokine therapy was comparedto the action of cytarabine (100 mg/kg daily for five consecutive days).Cytarabine is frequently used at this dose and schedule in preclinicalexperiments (43), but we also studied the effect of the drug in a pre-liminary therapy experiment in our AML model (fig. S4, D and E).Furthermore, the effect of F8-IL2 (30 mg of F8-IL2 every third day,four injections in total) in combination with cytarabine (100 mg/kgdaily for five consecutive days) was studied (Fig. 3, E and F). Admin-istration of F8-IL2, but not KSF-IL2 or cytarabine, significantly de-layed tumor progression up to day 27. In addition, the combinationof F8-IL2 with cytarabine led to long-lasting complete tumor eradica-tion in 40% of the treated mice.

In some cancer models, the antitumor activity of IL-2 is primarilymediated by T cells rather than by NK cells (42). For this reason, theeffect of F8-IL2 was studied also in an immunocompetent mouse mod-el (C57BL/6), bearing subcutaneously grafted C1498 tumors (~75 mm3).Immunofluorescence analysis of the established chloromas revealedintense staining with the F8 antibody and moderate staining with L19( F4Fig. 4A). Biodistribution experiments, performed as described above,showed a selective accumulation of F8-IL2 in the neoplastic lesions,with 3.1% injected dose per gram of tumor 24 hours after injectionand a tumor-to-blood ratio of 8.7 (Fig. 4B). In this setting, F8-IL2(30 mg every third day, three injections in total) mediated potent tu-mor growth retardation up to day 30. The combination of F8-IL2 andcytarabine (100 mg/kg daily for five consecutive days) led to completetumor eradication in 100% of the mice (Fig. 4, C and D).

In vivo depletion experiments, performed in C57BL/6J mice bear-ing subcutaneously grafted C1498 tumors, showed that the therapeuticactivity of F8-IL2 was completely conserved after depletion of CD4+

T cells, whereas tumor growth inhibition was lost as a result of NK cellor CD8+ T cell depletion (Fig. 4, F and G).

Clinical evaluation of F16-IL2 in combinationwith low-dose cytarabineIn an AML patient with rapidly progressing, generalized chloromadisease, an individual treatment was attempted using F16-IL2 in com-bination with low-dose cytarabine. The patient had previously experiencedmultiple relapses and had received allogeneic stem cell transplanta-tions from two different unrelated donors. A pretherapeutic 18-FDG-PET/computed tomography (CT) scan revealed multiple hypermetabolicthoracic and abdominal chloroma nodules, with standard uptake valuesof up to 14.1 ( F5Fig. 5). A lesion located in the hilum of the liver leading

Fig. 2. Splice isoforms of on-cofetal fibronectin (EDA andEDB) and tenascin-C are ex-

pressed in chloromas and colocalize with vasculature. (A) Sections ofchloromas from AML patients were analyzed by immunohistochemistryusing the clinical-stage F8, L19, and F16 antibodies, demonstrating thatEDA, EDB, and A1 of tenascin-C are expressed in chloroma specimens.Scale bars, 100 mm. (B) To show vascular colocalization, we performed im-munofluorescence using the F16 antibody (green), in combination with anantibody specific to vWF (red), as marker of blood vessels. Scale bar, 100 mm.DAPI, 4′,6-diamidino-2-phenylindole.





to cholestasis and a deep cervical massleading to difficulties in swallowing andvessel compression had to be simulta-neously irradiated. However, the patientpresented multiple other extramedullaryAML lesions in areas not targeted by ra-diotherapy. PET/CT images acquired onday 14 after therapy initiation with F16-IL2 (30 MIU of IL-2 equivalents intra-venously on day 1, 50 MIU on day 8) andlow-dose cytarabine (5 mg twice daily sub-cutaneously on days 1 to 10) showed anearly complete metabolic response ofboth the irradiated and nonirradiated le-sions after systemic treatment, which wasaccompanied by a partial morphologicalresponse in CT scans (Fig. 5). Notably,the clinical symptoms such as difficultiesin swallowing and restricted head mobil-ity improved shortly after systemic ther-apy was initiated, even before the firstapplication of radiotherapy.

DISCUSSION

The initial chemotherapy in AML com-prises a first phase of induction and asecond phase of consolidation. In mostof the patients, the induction treatmentleads to CR, defined as microscopic dis-appearance of leukemic disease along withthe return of normal hematopoiesis. How-ever, despite the introduction of more ef-ficacious consolidation regimens, a largeproportion of AML patients in CRwill sub-sequently experience relapses with poorprospects of long-term survival. A relapseis assumed to be the result of expansionof residual leukemic cells that have es-caped the initial chemotherapy. The anti-leukemic function of T cells and NK cellshas formed the background for the clinicaluse of IL-2, with the aim to eliminate resid-ual leukemia and hence reduce the relapserate in AML. Results of clinical trials usingIL-2 monotherapy in AML patients havebeen disappointing (44), but a recent phase3 study has demonstrated that postcon-solidation treatment with the combinationof histamine dihydrochloride and IL-2significantly prevents relapse in AML pa-tients (45).

There is a strong preclinical and clin-ical rationale suggesting that tumor-homingimmunocytokines can display a superioranticancer activity compared to nontar-geted IL-2 (9, 12). Vascular-targeting

Fig. 3. F8-IL2 selectively localizes to subcutaneoushumanAML tumors inmiceandcanpromote reductionin tumorprogressionaswell as complete tumoreradication incombinationwithcytarabine.HumanAMLcelllines NB4, HL60, and THP1 were subcutaneously implanted in nude mice. (A) Immunofluorescence analysis of theresulting tumors showed an intense staining with the F8 antibody and weak staining with L19 (red). Blood vesselswere costained using an anti-CD31 antibody (green). Scale bars, 100 mm. (B toD) Biodistribution experiments wereperformed by intravenous injection of radioiodinated F8-IL2 (black bars) or of the anti-lysozyme KSF-IL2 fusion pro-tein, usedasnegativecontrolof identicalmolecular format (whitebars). (B) InNB4xenografts (n=6), F8-IL2displayedtumor accumulationwith5.3% injecteddosepergram (ID/g) of tumor24hours after injectionanda tumor-to-bloodratioof18.8.HL60(n=3) (C)andTHP1(n=3) (D)xenograftsdisplayednotumor-targetingeffects.Nudemicebearingsubcutaneously grafted NB4 tumors (~40mm3) were treatedwith intravenous injections (n = 5) of either saline (×),F8-IL2 (▪) (30 mg, days 8, 11, 14, and 17), KSF-IL2 (□) (same dose and schedule), or cytarabine (▴) (100 mg/kg daily,days 8 to 12). Furthermore, the effect of F8-IL2 in combinationwith cytarabine (▵) was studied. (E and F) In addition,the combination of F8-IL2with cytarabine led to long-lasting complete tumor eradication in 40%of the treatedmice[*, significant for F8-IL2versus saline (P=0.0014)andKSF-IL2 (P=0.0025)afterday17; **, afterday15 for combinationversus saline (P = 0.0017) and KSF-IL2 (P = 0.0021), n = 5, two-tailed Student’s t test]. Data represent mean tumorvolumes± SD. Arrows depict days of treatmentwith immunocytokines (black) and cytarabine (gray). (G)Monitoringof the bodyweight showed that the combination treatment reduced average bodyweight, without exceeding thetoleratedbodyweight loss of 15%.Data represent averagepercent bodyweight relative to first day of therapy±SD.





immunocytokines have progressed to phase 2 clinical trials for thetreatment of patients with solid tumors (9, 12, 26) and have recentlybeen evaluated preclinically for the therapy of lymphomas (27). Here,we show that the clinical-stage F8 and F16 antibodies (and, to a lowerextent, L19) stain a considerable fraction of AML and ALL bone mar-row biopsies as well as AML chloromas in immunohistochemicalanalysis. We report the therapeutic effect of the immunocytokine F8-IL2, alone and in combination with cytarabine, in subcutaneous modelsof AML. F8-IL2 treatment promoted significant tumor growth retar-

dation and synergized with cytarabine, allowing the complete eradica-tion of tumors that could not be cured with cytarabine alone.

Complete leukemia eradication was only observed in the syngeneicimmunocompetent mouse model of AML, but not in immunodefi-cient mice bearing human leukemia cells, suggesting that T cells werenecessary for cancer eradication. In vivo depletion experiments indi-cated that the antitumor effect of F8-IL2 was mediated by both NKcells and CD8+ T cells, whereas CD4+ T cells did not appear to sig-nificantly contribute to the therapeutic action in the C1498 model.

Fig. 4. F8-IL2 treatment significantly re-duces tumor progression and can pro-mote complete tumor eradication incombination with cytarabine in subcu-taneous murine AML model. The murineAML cell line C1498 was subcutaneously im-planted in C57BL/6J mice. (A) Immuno-fluorescence analysis of the resulting tumorsshowed an intense staining with the F8 anti-body and weaker staining with L19 (red).Blood vessels were costained using an anti-CD31 antibody (green). Scale bars, 100 mm.(B) Biodistribution experiments were per-formed by intravenous injection of radio-iodinated F8-IL2 (black bars) or KSF-IL2,used as negative control of identical molec-ular format (white bars). F8-IL2 displayed3.1% injected dose per gram of tumor 24 hoursafter injection and a tumor-to-blood ratio of 8.7(n = 5). C57BL/6J mice bearing subcuta-neously grafted C1498 tumors (~75mm3) weretreated with intravenous injections (n = 5) ofeither saline (×), F8-IL2 (▪) (30 mg, days 7, 10,and 13), KSF-IL2 (□) (same dose and sched-ule), or cytarabine (▴) (100 mg/kg daily, days7 to 11). Furthermore, the effect of F8-IL2 incombination with cytarabine (▵) was studied.(C and D) The combination of F8-IL2 with cy-tarabine led to long-lasting complete tumoreradication in 100% of the treated mice [**,significant after day 11 for F8-IL2 versusKSF-IL2 (P = 0.0018) and saline (P = 0.0015);#, significant for combination group versusKSF-IL2 (P = 0.0024) and saline (P = 0.0016)after day 15AQ4 , n = 5, two-tailed Student’s t test].Data represent mean tumor volumes ± SD.Arrows depict days of treatment with immu-nocytokines (black) and cytarabine (gray). (E)Monitoring of the body weight showed thatthe combination treatment reduced averagebody weight, without exceeding the toler-ated body weight loss of 15%. Data representmean percent body weight relative to firstday of therapy ± SD. (F and G) In vivo immu-nodepletion experiments were performed inC57BL/6J mice bearing subcutaneously graftedC1498 tumors (n = 5). F8-IL2 promoted sig-nificant tumor growth retardation in unde-pleted (▪) and CD4+ T cell–depleted (○) mice,whereas animals with CD8+ T cell (•) or NK cell depletion (□) displayed atumor progression comparable to that of undepleted and saline-treated (×)mice. Data represent average tumor volumes ± SD. Arrows depict days of

treatment with immunocytokines (black) and antibodies for depletion(gray). (H) No body weight loss was observed. Data represent mean per-cent body weight relative to first day of therapy ± SD.





Leukemia cells appear to be particularly sensitive to the action ofantibody-based IL-2 delivery, because complete tumor eradicationswere never observed in the past, using IL-2–based immunocytokinesin other models of cancer (27, 33, 45–47). In a clinical perspective, it isimportant to consider that lymphocyte and NK cell counts are essen-tially normal in AML patients, which are otherwise often leukopenic(48). Thus, both NK cells and CD8+ T cells should be available forpromoting the anticancer effect of IL-2–based immunocytokines.

In the setting of compassionate use, a heavily pretreated AML pa-tient with rapidly progressing, generalized chloroma disease was treatedwith a combination of F16-IL2 and low-dose cytarabine. PET/CT im-ages before and on day 14 after therapy initiation revealed markedreduction of the AML lesions. These findings illustrate the potentialof the clinical application of immunocytokines in combination withcytarabine. In principle, such results could theoretically have beenachieved with cytarabine alone, yet in our opinion, the rapid and near-ly complete response to combination therapy in a patient who has failedmultiple lines of cytarabine-based chemotherapy in the past and whohas now received a very low dose of cytarabine is more supportive of asignificant contribution of the immunocytokine. Furthermore, beingafter allogeneic transplantation, the donor cellular immune systemmight have added to the efficacy of the immunocytokine. However,although the patient’s chronic skin graft-versus-host disease slightlyworsened upon therapy with F16-IL2, the most relevant side effectswere elevated body temperature and transient pain at the tumor sitesafter each application with otherwise good tolerability, suggesting atargeted therapeutic immune effect.

Bone marrow specimens of one AML patient showed positive stain-ing both at presentation and after CR because of induction therapy.Together with the observation that the bone marrow sample of anAML patient in aplasia was strongly stained by F8 and F16, this sug-gests that the antigen expression persists after chemotherapy (fig. S5).These findings are somewhat unexpected when taking into accountthe reports by Padró et al. (38), which show that AML patients enjoy-ing a CR after induction therapy exhibited a reduction of microvesseldensity to values comparable to the ones of control subjects. However,antigen persistence could reflect the high chemical stability of extra-cellular matrix components (such as fibronectin and tenascin splice iso-forms) generated during neovascularization processes. This feature islikely to favorably influence the development of armed antibody ther-apeutics, which can persist at the site of disease for several days.

A clear limitation of our study is the lack of therapy experimentsperformed in systemic disease, which involves the bone marrow. Weattempted to reproduce the promising targeting results that were ob-tained with chloroma models, in orthotopic mouse models of AMLwith florid proliferation of blasts in the bone marrow. Unfortunately,the models that we investigated so far did not express the EDA do-main of fibronectin, in contrast to what is observed in human AMLspecimens. In general, IL-2 immunocytokines have previously beenused to eradicate established neuroblastoma metastases in the bonemarrow (49). This observation indicates the use of immunocytokinesfor the therapeutic delivery of IL-2 to the diseased bone marrow in oth-er pathologies, such as leukemias.

F8-IL2 has not yet been studied in clinical trials, but F16-IL2 andL19-IL2 have been extensively studied in more than 200 patients withcancer (9, 12). F16-IL2 has been studied in two phase 1b trials in com-bination with paclitaxel or with doxorubicin (45). L19-IL2 is currentlybeing tested for the treatment of metastatic melanoma in a phase 2bstudy in combination with dacarbazine (26). It has also been studiedas monotherapy treatment for patients with renal cell carcinoma (50)and is completing a phase 1b trial in combination with gemcitabine inpatients with pancreas cancer. Both immunocytokines have shownpromising safety profiles and high combinability in patients (26, 50).

The incidence of AML increases with age, and there is an unmetmedical need for the treatment of elderly patients, who do not respondto approved therapeutic modalities and who do not tolerate aggressivechemotherapeutic regimens. The side effects of IL-2–based immuno-cytokines are generally mild, when these products are used at doses upto 67.5 MIU per week (26, 50). The management of patients becomesmore problematic, when high-dose IL-2 regimens are used, with re-peated administrations of >40 MIU doses to young patients in the in-tensive care setting (51).

The strong and selective antigen expression in acute leukemia, theemerging use of IL-2–based immunocytokines in clinical trials, as wellas the orthogonal profiles of side effects with F8/F16-IL2 and cytarabineprovide a rationale for the combined use of these agents for the treat-ment of AML patients (particularly those who are not eligible for bonemarrow transplantation or suffer from chloroma disease).

MATERIALS AND METHODS

Study designImmunohistological analysis of AML patient biopsies was performedwith the clinical-stage human monoclonal antibodies F8, F16, and

Fig. 5. PET/CT images of an AML patient with multiple chloroma man-ifestations treated with the immunocytokine F16-IL2 in combinationwith low-dose cytarabine. A 51-year-old female patient with refractoryAML after two allogeneic stem cell transplantations and rapidly progress-ing, disseminated extramedullary AML manifestations was treated withF16-IL2 (30 MIU intravenously on day 1, 50 MIU on day 8) and low-dosecytarabine (5 mg twice daily subcutaneously on days 1 to 10). 18-FDG-PET/CTimages were acquired before (day 0) and on day 14 after initiation of ther-apy. The two lesions marked with an arrow were irradiated simultaneouslyto systemic therapy because of local complications (cholestasis, swallowingdifficulties, and vessel compression). Clinical improvement started 1 day af-ter first infusion of F16-IL2 and before start of radiotherapy. A nearlycomplete metabolic response of irradiated and nonirradiated extra-medullary AML lesions could be documented as early as on day 14.





L19. The use of freshly frozen material was made necessary by the factthat these antibodies do not work in paraffin (36, 41), which preventedthe use of larger collections of formalin-fixed, paraffin-embedded spec-imens. Samples were collected at three medical centers (Muenster,Berne, and Kiel) for routine histological and cytological analyses. Semi-quantitative immunofluorescence was performed on the largest sampleset (n = 17), and the staining was additionally verified by immuno-histochemistry of consecutive sections of the same samples. Medianpatient age was 61 years (20 to 82 years). In case of AML, the studycohort represented the most frequent FAB subtypes; however, the sub-types M3, M6, and M7 were not available for analysis. The ALLsamples included two common ALL and two T-ALL. The bone mar-row of AML and ALL patients was highly infiltrated by leukemic blasts(median, 80%). Tumor-bearing mice were generated by subcutaneousinjection of AML cells to analyze the therapeutic effect of F8-IL2 (be-cause the F16 antibody, which showed the strongest staining in patientbiopsies, is not cross-reactive to mouse) alone and in combinationwith cytarabine. Initial mouse experiments (model setup, preliminarybiodistribution studies, and dose-finding experiments) were performedwith three mice per group to limit the use of animals. Mouse models,which were selected for therapy experiments, were tested in target-ing studies with n = 6 (NB4) and n = 5 (C1498). Therapy experi-ments were performed with five mice per group. The therapyendpoint was defined when tumors reached a set volume [>1000 mm3

(NB4) or 1200 mm3 (C1498)]. Animals with tumors of 20 to 100 mm3

on first day of therapy were included in experiments and staged tomaximize uniformity among the groups. The therapeutic effect ofF8-IL2 was studied in two mouse models, with two experiments per-formed in each model. The clinical application of F16-IL2 in combi-nation with cytarabine was tested on the basis of compassionate use.The preliminary clinical data shown here represent the finding in oneAML patient.

TissuesBone marrow core biopsy and bone marrow aspiration (iliac crest)were obtained for routine histological and cytological analyses. A frag-ment of each bone marrow biopsy was embedded in cryoembeddingmedium and immediately frozen at −80°C. Two sets of specimenswere analyzed. The larger set from Muenster consists of bone marrowbiopsies of 16 patients. The sample set from Berne and Kiel consists offour bone marrow biopsies from AML patients and two chloromabiopsies. Additional patient characteristics can be found in table S1.

Cell lines, animals, and xenograft modelsThe human AML cell line HL60 and the murine AML cell line C1498were purchased from the American Type Culture Collection. The hu-man AML cell lines NB4 and THP1 were obtained from the GermanResource Center for Biological Material. Cell lines were cultured ac-cording to the supplier’s recommendations. Six- to 8-week-old femaleCB17/lcr SCID and BALB/c nude mice were purchased from CharlesRiver Laboratories. Six- to 8-week-old female C57BL/6J mice werepurchased from Elevage Janvier. For the localized xenograft (chloroma)models, 107 HL60, NB4, or THP1 cells were subcutaneously injectedinto the flank of 8- to 10-week-old BALB/c nude mice. C1498 cells(106) were injected into the flank of 8- to 10-week-old C57BL/6J mice.All animal experiments were performed on the basis of project license(198/2008) administered by the Veterinäramt des Kantons Zuerichand approved by all participating institutions.

Antibodies and therapeutic agentsF8 is a human monoclonal antibody specific to the EDA domain offibronectin (34). The expression and characterization of the F8-IL2immunocytokine, as well as the control immunocytokine KSF-IL2,have previously been described (40). F16 is a tumor-targeting antibodyspecific to the domain A1 of human tenascin-C (32). L19 is a humanmonoclonal antibody specific to the EDB domain of fibronectin (35).KSF is specific to hen egg lysozyme and does not show any specificitytoward human antigens (42). All antibodies were biotinylated in theSIP format and carried a comparable number of biotin molecules.Cytarabine was purchased in solution from Sandoz.

ImmunofluorescenceTissue samples were snap-frozen, embedded in cryoembedding medi-um (Richard-Allan Scientific Neg-50; Thermo Scientific), and thenstored at −80°C. Consecutive tissue sections of 10-mm thickness wereprepared with a Microm HM 505N. Immunofluorescence was per-formed as previously described (31). For staining of the patient sam-ples from the Muenster set, the following primary antibodies wereused: biotinylated F8, L19, F16, or KSF (2 mg/ml) and polyclonal rabbitanti-human vWF (Dako). For the tumor and bonemarrow sections of themouse models, the following primary antibodies were used: biotinylatedF8, L19, F16, or KSF (2 mg/ml) andmonoclonal rat anti-CD31 antibody(1.6 mg/ml) (BD Biosciences). Detection of the biotinylated antibodieswas performed with streptavidin Alexa Fluor 594 (Invitrogen). As sec-ondarydetectionantibody for theanti-vWFantibody,AlexaFluor488 goatanti-rabbit (Invitrogen) antibody was used. The anti-CD31 antibodywasdetected with Alexa Fluor 488 donkey anti-rat (Invitrogen). The sampleswere analyzed on an Axioskop2mot plus microscope (Zeiss) with ZeissAxioVision v4.0 acquisition software.

Semiquantitative analysis of immunofluorescenceThe immunofluorescence stainings of leukemia patient biopsies weresemiquantitatively analyzed with ImageJ (http://rsb.info.nih.gov/ij/) bycomputing the percentage area of staining for each antibody. Thevalues were plotted as a boxplot (SigmaPlot).

ImmunohistochemistryImmunohistochemistry of leukemia patient samples was performed aspreviously described (29). Biotinylated F8, L19, and F16 (2 mg/ml)were used as primary antibodies. For the analysis of the Muenster sam-ples, KSF (2 mg/ml) was used as a negative control. For the Berne/Kielsamples, negative control was performed by omitting the primary anti-body. For detection, streptavidin–alkaline phosphatase complex (1:1000dilution) (Biospa) was used followed by subsequent reaction with thephosphate substrate Fast Red TR Salt (Sigma-Aldrich). Sections werecounterstained with hematoxylin solution Gill No. 1 (Sigma-Aldrich),mounted with Glycergel mounting medium (Dako), and analyzed onan Axiovert S100 TV microscope (Zeiss) with Zeiss AxioVision v4.0acquisition software.

Quantitative biodistribution studiesF8-IL2 and KSF-IL2 were radioiodinated with 125I, purified, and vali-dated for immunoreactivity as previously described (45). RadiolabeledF8-IL2 or KSF-IL2 (20 mg of antibody, 20 mCi per mouse) was injectedintravenously into BALB/c nude mice bearing subcutaneous NB4 (n = 6),HL60 (n = 3), or THP1 (n = 3) tumors, or C57BL/6 mice bearing sub-cutaneous C1498 (n = 5) tumors. Mice were sacrificed after 24 hours,





and the organs were excised and weighed. Radioactivity was measuredwith a Packard Cobra gamma counter. Radioactivity content of repre-sentative organs was expressed as percentage of the injected dose pergram of tissue (%ID/g ± SE).

Therapy studies in localized xenograft (chloroma) modelsNB4 cells (107) were injected subcutaneously into the flank of 6- to8-week-old female BALB/c nude mice. C1498 cells (106) were injectedsubcutaneously into the flank of 6- to 8-week-old female C57BL/6mice. When tumors were established (20 to 100 mm3), mice werestaged to maximize uniformity among the groups (n = 5) and injectedinto the lateral tail vein with either saline, 30 mg of F8-IL2, 30 mg ofKSF-IL2, cytarabine (100 mg/kg), or a combination of F8-IL2 andcytarabine (at same dosage). Treatment schedule for the immunocy-tokines was every third day for four and three injections, respectively.Cytarabine was administered daily for 5 days. The mice were mon-itored daily, and tumor growth was measured every second day witha digital caliper using the following formula: volume = length × width ×width × 0.5. Animals were sacrificed when the tumor reached a volumeof >1200 mm3. The data were displayed as average values ± SD.

In vivo depletion of NK cells, CD4+, and CD8+ T cellsSubcutaneous C1498 tumors were established in C57BL/6 as describedabove. Tumor-bearing mice (n = 5) were injected intraperitoneallywith either saline, anti–asialo GM1 (30 ml) (Wako Chemicals), anti-CD4 (Bio X Cell), or anti-CD8 (Bio X Cell) antibodies on days 6, 9,12, and 15. F8-IL2 (30 mg) was administered intravenously on days 7,10, and 13. The mice were monitored as described above. The datawere displayed as average values ± SD.

Treatment of a chloroma patientA female patient was diagnosed with complex karyotype AML FABM1 in February 2007 at 45 years of age. Upon being refractory to in-duction chemotherapy with cytarabine and daunorubicin (7 + 3) andhigh-dose cytarabine and mitoxantrone (HAM), she received un-related donor allogeneic stem cell transplantation after conditioningwith fludarabine, cytarabine, amsacrine, cyclophosphamide, anti-thymocyteglobulin, and 2 × 2 Gy total body irradiation in October 2007, where-upon she enjoyed a first CR. In July 2008, she experienced AML re-lapse, and therapy with low-dose cytarabine (persistent AML), high-dosecytarabine, and an allogeneic stem cell boost resulted in a second CR,which lasted more than 2.5 years. However, in May 2011, a secondAML relapse was diagnosed. Although chemotherapy with HAMand a donor stem cell boost were ineffective, allogeneic stem cell trans-plantation from a different unrelated donor led to a short-lasting CRuntil January 2012, when the third relapse occurred. Chemotherapywith FLAG-IDA and an additional donor stem cell boost resultedin another phase of CR. In November 2012, however, extramedullaryAML manifestations were diagnosed and confirmed histologically inboth mammary glands (whereas the bone marrow was still leukemia-free with stable donor cell chimerism) and palliative local radiotherapy(37 Gy) was performed. Three months later, multiple chloroma nod-ules occurred in the abdominal skin and were again treated with localradiotherapy (36 Gy) until the beginning of May 2013. Three weekslater, intra- and extrahepatic cholestasis led to the diagnosis of rapidlyprogressing, disseminated chloroma disease with manifestations in theleft deep cervical area, in the mediastinum, in the hilum of the liver, inthe mesenterium, in the hilum of the right kidney, in the manubrium

sterni, in the transverse process of the ninth thoracic vertebra, and sub-cutaneously in the left gluteal area. In this desperate situation, our pa-tient received the immunocytokine F16-IL2 (30 MIU intravenously onday 1, 50 MIU on day 8) and low-dose cytarabine (5 mg twice dailysubcutaneously on days 1 to 10). Ethical approval for this individualcompassionate use therapy was obtained from the joint ethical boardof the University of Muenster and the locoregional physician’s cham-ber of Westfalen-Lippe. Written informed consent was obtained inaccordance with the Declaration of Helsinki. A phase 1b study withF16-IL2 in combination with chemotherapy is running at multiplecenters (NCT01134250, NCT01131364) and had shown that dosesup to 55 MIU can be safely administered to patients (52).

Statistical analysisThe staining of bone marrow biopsies of AML patients with F8, F16,and L19 was analyzed compared to the negative control antibody KSF.Values exceeding the 95% confidence interval (average + 2 × SD) ofKSF (3.4% for AML samples; 2.6% for ALL samples) were consideredpositive.

In therapy experiments in mice, the differences in tumor volumebetween therapeutic groups were compared with the two-tailed Student’st test with a = 0.05. Taking the Bonferroni correction into account,significance was determined when P ≤ 0.003.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/5/201/201ra118/DC1Materials and MethodsFig. S1. Splice isoforms of oncofetal fibronectin (EDA and EDB) and tenascin-C are expressed inbone marrow biopsies of acute leukemia patients.Fig. S2. Splice isoforms of oncofetal fibronectin (EDA and EDB) and tenascin-C are expressed inbone marrow and chloroma biopsies of acute leukemia patients.Fig. S3. EDA is not expressed in orthotopic models of leukemia, despite disease progression inblood and bone marrow.Fig. S4. Treatment of subcutaneous NB4 tumors with F8-IL2 promotes tumor growth retardation.Fig. S5. Antigen expression persists in bone marrow biopsies of AML patients in completeresponse and aplasia.Table S1. Patient characteristics of the sample set from Muenster.Table S2. Patient characteristics of the sample set from Berne and Kiel.

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Funding: This work was supported by the Swiss National Science Foundation, the ETH Zuerich,the European Union (ADAMANT Project and IMMOMEC Project), the Swiss Cancer League, theSwiss Bridge Foundation, and the Stammbach Foundation. Author contributions: K.L.G. con-ceived and performed the experiments and wrote the manuscript; C.S. conceived theexperiments, performed initial immunohistochemical experiments, and contributed to themanuscript; L.G. supplied F16-IL2 for clinical application; K.F. produced the fusion proteins





F8-IL2 and KSF-IL2 and helped in the generation of the orthotopic model; T.P. and W.K.collected, characterized, and provided human leukemia specimens and corrected the manu-script; W.E.B. collected, characterized, and provided human leukemia specimens andcontributed to the manuscript; D.N. conceived the experiments, supervised the experimentalwork, and wrote the manuscript. Competing interests: D.N. is a co-founder and shareholderof Philogen. L.G. is the head of clinical operations at Philogen (http://www.philogen.com), thecompany that owns the F8, F16, and L19 antibodies. The other authors declare that they haveno competing interests.

Submitted 13 March 2013Accepted 26 June 2013Published 4 September 201310.1126/scitranslmed.3006221

Citation: K. L. Gutbrodt, C. Schliemann, L. Giovannoni, K. Frey, T. Pabst, W. Klapper,W. E. Berdel, D. Neri, Antibody-based delivery of inteleukin-2 to neovasculature has potentactivity against acute myeloid leukemia. Sci. Transl. Med. 5, 201ra118 (2013).





Abstracts

One-sentence summary: Neovascular structures in acute myeloid leukemia can be targeted using a cognateantibody fused to human interleukin-2.

Editor’s Summary:The Blood Mobile

For solid tumors, new blood vessel formation—angiogenesis—is thought to be critical to bring oxygen and nu-trients to all parts of the tumors, and targeting these new vessels has long been a focus of cancer therapy devel-opment. However, angiogenesis-targeting therapy has been relatively neglected in the context of blood-bornetumors like leukemias. Now, Gutbrodt et al. find that targeting neovascular structures found in the bone marrowof patients with acute myeloid leukemia (AML) has activity against blood-borne cancers as well.The authors observed that AML patients have increased blood vessel density in the bone marrow, and that

these vessels could be stained by clinical-stage human antibodies against tumor angiogenesis markers. Theythen took this observation into mouse models and found that if they fused interleukin-2 to the targetingantibody, they could inhibit AML progression. The effect was further enhanced by combination with thechemotherapeutic cytarabine. Indeed, this effect was long-lasting in immunocompetent mice and wasmediated by both natural killer and CD8+ T cells. Early results from clinical trials support these results inhuman patients, although further trials are needed to confirm efficacy.





antibody-based delivery of inteleukin-2 to neovasculature ...¤mie_science.pdf · immunochemical...

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