Targeting Interleukin-2 to the Bone Marrow Stroma for ...Target expression in pretherapeutic bone marrow TnC-A1 was expressed with a vascular and stromal pattern of staining in the

Research Article

Targeting Interleukin-2 to the Bone MarrowStroma for Therapy of Acute Myeloid LeukemiaRelapsing after Allogeneic Hematopoietic StemCell TransplantationChristoph Schliemann1, Katrin L. Gutbrodt2, Andrea Kerkhoff1, Michele Pohlen1,Stefanie Wiebe1, Gerda Silling1, Linus Angenendt1, Torsten Kessler1, Rolf M. Mesters1,Leonardo Giovannoni3, Michael Sch€afers4, Bianca Altvater5, Claudia Rossig5,Inga Gr€unewald6, EvaWardelmann6, Gabriele K€ohler6,7, Dario Neri2, Matthias Stelljes1, andWolfgang E. Berdel1

Abstract

The antibody-based delivery of IL2 to extracellular targetsexpressed in the easily accessible tumor-associated vasculaturehas shown potent antileukemic activity in xenograft and immu-nocompetent murine models of acute myelogenous leukemia(AML), especially in combination with cytarabine. Here, wereport our experience with 4 patients with relapsed AML afterallogeneic hematopoietic stem cell transplantation (allo-HSCT),who were treated with the immunocytokine F16-IL2, in combi-nation with low-dose cytarabine. One patient with disseminatedextramedullary AML lesions achieved a complete metabolicresponse identified by PET/CT, which lasted 3 months. Two of3 patients with bone marrow relapse achieved a blast reduction

with transientmolecular negativity. One of the 2 patients enjoyeda short complete remission before AML relapse occurred 2months after the first infusion of F16-IL2. In line with a site-directed delivery of the cytokine, F16-IL2 led to an extensiveinfiltration of immune effector cells in the bone marrow. Grade2 fevers were the only nonhematologic side effects in 2 patients.Grade 3 cytokine-release syndrome developed in the other 2patients but was manageable in both cases with glucocorti-coids. The concept of specifically targeting IL2 to the leukemia-associated stroma deserves further evaluation in clinical trials,especially in patients who relapse after allo-HSCT. Cancer Immu-nol Res; 3(5); 547–56. �2015 AACR.

IntroductionTherapeutic approaches interfering with tumor angiogenesis

have become fully incorporated into clinical practice nowadays(1). In addition to antiangiogenesis, a second, conceptuallydifferent strategy, "vascular targeting," takes advantage of tumorvessels for therapeutic purposes and aims at the selective phar-macodelivery of therapeutic payloads (e.g., drugs, cytokines,radionuclides) to the tumor site by their conjugation to carrierantibodies, which specifically home to tumor-associated vascu-lature (2–5). The approach benefits from the intrinsic accessibilityof vascular targets and bypasses many of the hurdles associated

with the targeting of cancer cell markers, such as physical andkinetic barriers of bulky tumors or antigen heterogeneity amongtumor cells. Furthermore, whereas cancer cell surface proteins areinmany cases shared by normal cells, vascular and stromal targetsare available, allowing a clear-cut discrimination between healthyand neoplastic tissues (2, 3). Although it was initially assumedthat vascular-targeting antibodies would mainly be relevant forsolid tumors, we recently discovered that well-characterized vas-cular targets are abundantly expressed in hematologic malignan-cies, such as Hodgkin and non-Hodgkin lymphomas, allowingthe antibody-mediated deposition of bioactive payloads at thelymphoma site (6, 7).

In 2000, we reported that acute myelogenous leukemia (AML)is associated with extensive neovessel formation in the bonemarrow (8). In an attempt to evaluate a vascular-targeting strategyin AML, we have recently shown that the alternatively splicedextra-domains A (EDA-Fn) and B (EDB-Fn) of fibronectin andthe extra-domain A1 of tenascin-C (TnC-A1) are abundantlyexpressed in AML bone marrow and extramedullary AML lesions(5). These markers are almost exclusively found in the tumor-associated vasculature and stroma while being virtually absent innormal organs and have been shown to be suited for site-specificpharmacodelivery approaches (2, 3, 9, 10). In immunocompro-mised and immunocompetent mouse models of myelosarcoma-type AML, the antibody-mediated delivery of the proinflamma-tory cytokine IL2 to the leukemia-associated vasculature led to

1Department of Medicine A, Hematology and Oncology, UniversityHospitalMuenster,Muenster,Germany. 2DepartmentofChemistryandApplied Biosciences, ETH Zurich, Zurich, Switzerland. 3Philogen SpA,Siena, Italy. 4Department of Nuclear Medicine, University HospitalMuenster, Muenster, Germany. 5Department of Pediatric Hematologyand Oncology, University Children's Hospital Muenster, Muenster,Germany. 6Gerhard-Domagk-Institute for Pathology, University Hos-pital Muenster, Muenster, Germany. 7Institute of Pathology, GeneralHospital Fulda, Fulda, Germany.

Corresponding Author: Wolfgang E. Berdel, University Hospital Muenster,Albert-Schweitzer-Campus 1, D-48149 Muenster, Germany. Phone: 49-251-8347587; Fax: 49-251-8347588; E-mail: [email protected]

doi: 10.1158/2326-6066.CIR-14-0179

�2015 American Association for Cancer Research.

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substantial leukemia growth retardation in a process mediated byCD8þ T cells and natural killer (NK) cells, whereas equivalentdoses of nontargeted IL2 were ineffective (5). Most importantly,the combination of the immunocytokine with cytarabine pro-moted complete leukemia eradications.

Here, we report the use of the immunocytokine F16-IL2, whichmediates the selective delivery of human IL2 to TnC-A1, in com-bination with low-dose cytarabine (LDAC) in heavily pretreatedAML patients with relapsed or refractory disease after allogeneichematopoietic stem cell transplantation (allo-HSCT).

Case reportsPatient 1was awomandiagnosedwith complex karyotypeAML

French-American-British classification (FAB) M1 in February2007 at 45 years of age. Her detailed medical history has beendescribed previously (5). Briefly, in May 2013, the patient ulti-mately presented with rapidly progressing, disseminated myelo-sarcoma (chloroma) nodules after multiple lines of intensivetherapy, including two unrelated donor allo-HSCTs, while thebone marrow was free of leukemia. [18F]2-fluoro-2-deoxy-D-glucose PET/CT revealed hypermetabolic manifestations in theleft deep cervical area, in the mediastinum, in the hilum of theliver (leading to cholestasis), in themesentery, in the hilum of theright kidney, in the sternum, in the transverse process of the 9ththoracic vertebra, and subcutaneously in the left gluteal area.

A 50-year-old woman (patient 2) had a diagnosis of NPM1-mutated, normal karyotype AML FAB M1 in August 2010. Induc-tion therapy with two cycles of high-dose cytarabine and mitox-antrone (HAM) resulted in a first complete remission (CR) andwas followed by consolidation with 6-thioguanine, cytarabine,and daunorubicin (TAD) and maintenance therapy according tothe Acute Myeloid Leukemia Cooperative Group 2008 protocol.In June 2011, the patient's first AML relapse occurred, and sheunderwentmatched related donor allo-HSCT, leading to a secondCR. Ten months later, the second AML relapse occurred, andanother allo-HSCT from a matched unrelated donor was per-formed after chemotherapy with amsacrine, fludarabine, andhigh-dose cytarabine (FLAMSA) and conditioning with melpha-lan, busulfan, fludarabine, and antithymocyte globulin (ATG).The second allo-HSCT resulted in another CR, but a third AMLrelapse was diagnosed 9 months later.

Patient 3 was a 53-year-old man diagnosed with AML second-ary to chronic myelomonocytic leukemia (CMML). CMML wasfirst diagnosed in August 2011 and was treated with azacytidineuntil August 2012, when the disease transformed to AML.Althoughhewas refractory toHAM induction therapy, he receivedallo-HSCT from a matched unrelated donor in December 2012after conditioning with melphalan, fludarabine, ATG, and 8-Gytotal body irradiation (TBI) and achieved a CR that lasted 6months. However, AML relapse was diagnosed in July 2013.

In January 2007, NPM1-mutated AML FAB M5 with normalkaryotype was diagnosed in patient 4 at the age of 39 years. Afterinductionwith two courses of daunorubicin and cytarabine (DA),he achieved a CR, followed by consolidation with three cycles ofhigh-dose cytarabine. InMay 2011, the first AML relapse occurred(now FLT3-ITD positive), and the patient underwent allo-HSCTwith a matched unrelated donor after chemotherapy with HAMand conditioningwith cyclophosphamide, ATG, and12-Gy TBI inJuly 2011, leading to a second CR of 2 years. In September 2013,NPM1-positive, FLT3-ITD–negative AML relapse was diagnosedwith the karyotype 46,XY,t(2;3)(q?33;?),t(5;6)(q1?3;p15)[7]/46,

XY[13]. At that time, the patient was reluctant to receive intensivechemotherapy followed by a second allo-HSCT.

Patients and MethodsPatients and treatment

From June 2013 to December 2013, 4 AML patients whorelapsed after multiple chemotherapies and one (n ¼ 2) or two(n ¼ 2) allo-HSCTs were treated in a compassionate-use settingwith F16-IL2 and LDAC at the University Hospital Muenster,Germany. The joint ethical board of the University of Muensterand the regional Physician's Chamber of Westfalen-Lippe wasconsulted for each individual case.Written informed consent fromeach patient was obtained in accordance with the Declaration ofHelsinki. F16-IL2, consisting of the human anti–TnC-A1 antibodyF16 fused to human IL2, was provided by Philogen. F16-IL2 wasadministered weekly as a 3-hour intravenous infusion. Patientsreceived 30Mio IU IL2 equivalents on thefirst infusion and 50MioIU in the following infusions when the first was well tolerated.LDAC was administered s.c. on days 1 to 10 at 5 mg twice daily.With the exception of patient 1, who required low-dose systemicsteroids for chronic skin graft-versus-host disease (GVHD),noneofthe other 3 patients received immunosuppressive medicationwhen post–allo-HSCT AML relapse was diagnosed and treatmentwith F16-IL2 and LDAC was initiated. A phase Ib study with F16-IL2 in combination with chemotherapy in patients with solidtumors is ongoing and has shown that doses up to 70 million IUcan be safely administered (NCT01134250 and NCT01131364).

Histochemical analysesFor analysis of TnC-A1 expression, cryosections of prethera-

peutic bone marrow biopsies were stained with biotinylated F16as described (5). Antibody detection was performed with strepta-vidinAlexa Fluor 488 (Invitrogen). For analysis of in vivo targeting,F16-IL2 bound to its target in situ was detected in posttherapeuticbone marrow specimens using the labeled streptavidin–biotin(LSAB) method. Sections of formalin-fixed paraffin-embeddedbonemarrowbiopsieswere prepared, followed by antigen retriev-al in citrate buffer (pH 6, 30 minutes, 99�C). Sections wereincubatedwith a rabbit anti-human IL2 antibody (1:125; Abcam)overnight at 4�C. Visualization was performed using Dako REALDetection System/AP following the manufacturer's instructions.Samples were analyzed on a Nikon Eclipse 50i microscope withthe NIS-Elements 4.13 acquisition software (Nikon).

Flow cytometryLymphocyte phenotypes were determined using fluorescence-

conjugated mAbs against CD3, CD4, CD8, CD16, CD19, CD56,and TCR gd (all from BD Pharmingen). For each sample, 20,000cells were analyzed with FACS Canto and FACS Diva Software.Intracellular FoxP3 expression was analyzed with the anti-humanFoxP3 staining Kit according to the manufacturers' recommenda-tions (eBioscience). The percentage of CD25hi/FoxP3þ cells wasdetermined after gating on CD3þ/CD4þ cells.

Bone marrow chimerismChimerism analyses were performed as previously described

using a semiquantitative PCR approach based on the amplifi-cation of short tandem repeat markers (11). Briefly, genomicDNA for multiplex PCR of microsatellite markers was extracteddirectly from mononuclear cells. Nine tetra-nucleotide micro-satellite regions were coamplified with dye-labeled primers

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using the AmpFLSTR Profiler PCR amplification Kit (AppliedBiosystems).

ResultsTarget expression in pretherapeutic bone marrow

TnC-A1 was expressed with a vascular and stromal pattern ofstaining in the bone marrow of all 3 patients with bone marrowAML involvement (Fig. 1A), providing the molecular basis for anF16-based targeting approach. In patient 1, only paraffin-embed-ded chloroma material was available, which could not be ana-lyzed for TnC-A1 expression, because the F16 antibody does notwork in paraffin-embedded tissues (12). However, we have pre-viously observed that extramedullary AML lesions abundantlyexpress TnC-A1 (5).

In vivo targeting of F16-IL2Posttherapy bonemarrowbiopsieswere available for patients 2

and 4 to analyze the in vivo–targeting capabilities of F16-IL2.Using an anti-IL2 antibody to detect F16-IL2 bound to its target insitu, IL2 signals decorating the extracellularmatrix were detectablein the bone marrow 6 days after the last infusion in patient 2, butnot in pretherapeutic bonemarrow (Fig. 1B), suggesting that F16-IL2 has efficiently targeted TnC-A1 in vivo and persisted for severaldays. On day 42, however, 3 weeks after the last infusion of F16-IL2, stromal IL2 signals were not observed any more (Fig. 1B,inset). In patient 4, no significant F16-IL2 signals could beobserved 6 days after infusion.

Clinical courseThe most impressive response was observed in patient 1 with

disseminated extramedullary AML. Shortly after the first infusionof F16-IL2, thepatient experiencedpain in the cervical and the rightupper abdominal chloroma lesions, suggesting a site-specific tar-

geting of the cytokine. The palpable tumors became softer andstarted to shrink on the following day, and swallowing problemsdue to the cervical mass resolved within 48 hours, while headmobility improved. All these signs of activity occurred before thebeginning of additional radiotherapy,whichwas initiated onday 4to ensure that the twomost critical chloroma lesions (cervicalmassand liver hilum)were efficiently treated (23.4Gy). On day 14 (i.e.,after two infusions of F16-IL2), a complete metabolic responseof all irradiated and nonirradiated AML lesions could be observ-ed in PET/CT, accompanied by a partial morphologic response(Fig. 2A). Although the first three infusions of F16-IL2 werewell tolerated, with transient fevers of 2 to 3 days after infusion,pain at sites of chloroma, and slight signs of skin and liver GVHDworsening, thepatient had tobehospitalized3days after the fourthadministration of the fusion protein (day 25) due to prolongedhigh fevers escalating to 40.5�C, respiratory distress, and increasingskinGVHD(stage3, erythroderma).ACT scan revealedpulmonaryinfiltrates in the middle lobe and bilateral pleural effusions.Because clinical and laboratory findings were compatible withcytokine release associated with a macrophage activation syn-drome, with elevation of ferritin levels (15,575 mg/L) on day 28,hypofibrinogenemia (81 mg/dL, 263 mg/dL before F16-IL2), andelevated IL6 levels (32 pg/mL, 6-fold increase frombaseline), high-dose corticosteroids were administered in addition to broad-spec-trum antimicrobials. The patient's fever resolved within 48 hours,her other clinical symptoms improved within the following days,and ferritin levels decreased to 2806 mg/L within 2 weeks. She wasdischarged on day 39. The patient did not receive the plannedsecond cycle of therapy but continued treatment with a reduceddose of 30 Mio IU of F16-IL2 in longer intervals of 3 weeks on anoutpatient basis. She resumed normal activity with a fair quality oflife (Karnofsky index of 80%–90%), no clinical signs of chloromadisease, and preexisting skin GVHD that was manageable with

C

A

B

IL2

IL2

Patient 2 Patient 3 Patient 4

TnC-A1 DAPI TnC-A1 DAPI TnC-A1 DAPI

Pretherapeutic BM Posttherapeutic BM

Figure 1.Expression of TnC-A1 in the bone marrow (BM) and in vivo targeting of F16-IL2. A, pretherapeutic stromal expression of TnC-A1 (green) could be detected in allpatients with bone marrow involvement of AML using biotinylated F16(SIP) antibodies (�200). B, the presence of F16-IL2 in posttherapeutic bone marrowwas highlighted using an antibody specific to human IL2 (�400). In patient 2, extracellular IL2 signals couldbedetected in situ6days (but not 3weeks, inset) after thelast infusion, suggesting successful targeting and on-target persistence of the immunocytokine for several days in vivo. Stromal IL2 signals were not detectablein pretherapeutic bone marrow that was not exposed to F16-IL2 treatment and used as control. C, schematic representation of the dimeric scFv–huIL2 fusionprotein F16-IL2 in diabody format.

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local and oral methylprednisolone. However, in September 2013,almost 4 months after the first infusion of F16-IL2, a follow-upPET/CT revealed disseminated recurrence of chloromas. Treatmentwas intensified to weekly applications of 50 Mio IU F16-IL2 incombination with LDAC; however, the patient did not respond asecond time. Because she expressed her distinct wish for furthertherapy, she was offered intensive chemotherapy with sequentialhigh-dose cytarabine and idarubicin. Unfortunately, the patientdieddue to infectious complications in the phaseofneutropenia inNovember 2013 on day 176.

The other patients displayed "classical"medullary AML. Patient2 showed a 50% to 60% bone marrow infiltration before treat-ment. After two infusions of the immunocytokine, bone marrowblasts as determined by cytologic analyses were reduced to 6% onday 14, and were further reduced to <5% on day 28, after fouradministrations of F16-IL2.Microscopic evaluation revealed ther-apy-induced extensive infiltration of activated lymphocytes, somewith blast-like morphology in the bonemarrow at the same time.The reduction ofmyeloid blasts was paralleled by a disappearanceof mutated NPM1 in PCR analyses and by an increase of bonemarrow donor cell chimerism from 63% to 98% (Fig. 3A). Nosignificant leukemia infiltration was detected in flow-cytometricanalyses (0.81% and 0.98% CD117þ blasts on days 14 and 28,respectively; 23.85% at baseline). Each infusion of the immuno-cytokine led to fever of up to 39.0�C,which typically lasted 2 days,but no other symptoms of cytokine release developed. Becausethere was no evidence of residual leukemia at themolecular level,therapy with F16-IL2 was paused for 2 weeks to allow regener-ation of normal hematopoiesis. By day 42, however, neutrophiland thrombocyte counts did not regenerate and AML progressedrapidly again. Treatment was resumed with higher doses of F16-IL2 [50Mio international units (IU) on day 1, 70Mio IE on days 8

and 15] in combinationwith LDAC,which, however, did not leadto a second response. The patient expressed a clear wish for furthertherapy, and she received a third allo-HSCT 3 months aftertreatment initiation with F16-IL2. This resulted in a short CRwith incomplete platelet recovery (CRp) of approximately 2months, until AML relapse ultimately occurred in February 2014.

Patient 3 presented with highly proliferative AML relapse inperipheral blood (4,900 leukemic blasts/mL) and bone marrowinfiltration of 70%. Treatment with F16-IL2 (30 Mio IU) andLDAC led to a blast reduction in peripheral blood (1,080/mL)within 1 week (Fig. 3B). However, the patient had to be hospi-talized after the first dose of F16-IL2 due to fevers (39.7�C), chills,nausea, and a reduced general condition. He was treated withbroad-spectrum antibacterials, but it was again suspected that thefevers weremore likely due to noninfectious hyperinflammation/cytokine release. Indeed, signs of capillary leakage developedwithin the next few days with generalized edema, weight gain(8 kg), and mild dyspnea. Laboratory findings showed elevatedlevels of IL6 (139 pg/mL, 10-fold increase from baseline), hyper-ferritinemia (11,600 mg/L), and a transient increase in creatinine(2.1 mg/dL). Fevers resolved by day 9 after 4 days of glucocorti-coids. Creatinine normalized within 4 days, the patient's weightreturned to baseline by day 13, ferritin levels decreased to 3,888mg/L, and he was discharged on day 15. F16-IL2 was not admin-istered on days 8, 15, and 22. In a phase of rapid leukemiaprogression in peripheral blood, the treatment was resumed onday 29 at 15Mio IU F16-IL2 in an outpatient setting and was welltolerated at this time. Five days later, however, the patient had asevere accident at home and died due to intracerebral hemorrhagein the hospital shortly after.

Patient 4 presented with at least 10% leukemic blasts (focallymore) in the bone marrow and 2% in peripheral blood.

Baseline Day 14

A BFigure 2.Rapid response to F16-IL2 and LDAC indisseminated extramedullary AML. A,the figure shows axial 18-FDG-PET/CTscans obtained at baseline beforetreatment initiation and 14 daysafter the first infusion (patient 1).Pretherapeutic images showdisseminated hypermetabolicmyelosarcomas (chloromas; SUVmax

14.1) in the left deep cervical area, inthe mediastinum, in the hilum of theliver, in the mesentery, in the hilum ofthe right kidney, in the manubrium ofthe sternum, in the transverse processof the 9th thoracic vertebra, andsubcutaneously in the left gluteal area.The two clinically most critical lesions(left cervical and hilum of the liver)were irradiated between day 4 andday 24 with a cumulative dose of 23.4Gy in addition to systemic therapy.A complete metabolic response ofirradiated and all nonirradiated AMLmanifestations occurred within 14days of treatment initiation, whichwas paralleled by a good partialmorphologic remission. B, signs of skinGVHD, which occurred during therapywith F16-IL2 in this patient.

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Pretherapeutic bone marrow donor cell chimerism decreased to83%. Peripheral blasts disappeared within the first few days oftreatment with F16-IL2 and LDAC, and the first bone marrowevaluation on day 14 revealed a reduction of blasts to approxi-mately 2% (CD33þ/CD117þ cells on flow cytometry, 12.3% atbaseline; Fig. 3C). Microscopic quantification showed�5%mye-loid leukemic blasts and an extensive infiltration with activatedlymphocytes. In molecular and conventional cytogenetic analy-ses, the markers NPM1, t(2;3) and t(5;6), were undetectable, andbone marrow donor cell chimerism increased to 96% to 97%.Thrombocyte counts increased to 157,000/mL, and neutrophilcounts improved to 2,214/mL after the first cycle (Fig. 3C), therebymeeting the criteria for a CR. However, after two cycles (2monthsafter the first infusion of F16-IL2), the patient experienced a bonemarrow relapse with 15% blasts and reappearance of the molec-ular and cytogenetic abnormalities. The patient received twoinfusions of donor-derived lymphocytes without response andunderwent a second matched unrelated alternative donor allo-HSCT in February 2014, but eventually died from infectiouscomplications in the recovery time following transplantation.

ToxicitiesAll toxicities are summarized in Table 1. The most frequent

adverse events were fever, chills, and fatigue, which occurred in allpatients. IL2-related infusion-associated hypotension was notobserved. Tumor pain as observed in the patient with chloromais an uncommoneffect of recombinant IL2 that could be related toF16-mediated targeting of the cytokine. In patients 2 and 4, feverstypically occurred either toward the end of the infusion or shortlyafter, regularly resolved by day 2 or 3, andwere easilymanageableat home with acetaminophen. In these patients, no other relevant

nonhematologic toxicities occurred (apart frommild skin GVHDin patient 2).

However, theother 2patientsbecame significantly ill after thefirstinfusionof 30Mio IUF16-IL2 (patient 3)or after the fourth infusionof 50 Mio IU F16-IL2 (patient 1), respectively. Both patients devel-oped symptoms of the cytokine-release syndrome described above,with laboratory features compatible with macrophage activationsyndrome, leading to hospitalization in both cases. The syndromecould be effectively treated with glucocorticoids. No anticytokineantibodies, mechanical ventilation, or vasopressor support wererequired. Nonhematologic grade 4 events were not observed.

Immunologic effects of F16-IL2Circulating lymphocytes in peripheral blood significantly

increased to levels 2 to 4 times as high as baseline levels by day11 in all patients. Therapy-induced changes of peripheral lym-phocyte subpopulations in patients 2 and 4 are shown in Fig. 4A.CD8þ lymphocytes peaked early after treatment initiationbetween days 8 and 11 with a 5.7-fold increase to 4,640/mL inpatient 2 and a 2-fold increase to 747/mL in patient 4. In contrast,peripheralNKcells showedamoreor less steady risewith repeatedadministrations of F16-IL2 to values as high as 3,567/mL and1,303/mL, respectively—a 17.7-fold and 16.5-fold increase frombaseline. At peak levels, NK cells accounted for more than 50% ofcirculating lymphocytes. There were no significant changes inCD19þ B cells. Interestingly, activated lymphocytes and lympho-cytes with large granular morphology could be readily detected inperipheral blood (Fig. 4B).

The most prominent changes were observed in the bonemarrow. F16-IL2 led to a massive accumulation of lymphocytesin the bone marrow (Fig. 4C and D). Whereas lymphocytes

A B CCells/µL

Cells/µL

Cells/µL

Days Days Days

Days Days

Thrombocytes(x10

3 /µL)

Thrombocytes(x10

3 /µL)

Thrombocytes(x10

3 /µL)

Bonemarrowcells(%)

Bonemarrowcells(%)

Figure 3.Clinical response to F16-IL2 and LDAC in patients with AML bone marrow involvement. Top, peripheral blood cellular counts, specifically the development of whiteblood cell (WBC) counts, peripheral blast counts, absolute neutrophil counts (ANC), absolute lymphocyte counts (ALC), and thrombocytes during therapywithF16-IL2 andLDAC in patients 2 (A), 3 (B), and4 (C).Arrows signify infusions of F16-IL2 at thedose indicated above (�106 IU IL2 equivalents); days of application ofLDAC (2� 5 mg/day s.c.) are marked with black bars. Bonemarrow blast counts as determined by flow-cytometric analyses and bone marrow donor cell chimerismlevels from aspirates taken at the time points indicated are depicted in the bottom plots (no posttherapy bone marrow aspirates were available in patient 3).






accounted for 15% to 20%of cells in pretherapeutic bonemarrowaspirates, lymphocytic infiltration of the bone marrow wasapproximately 80% in patient 2 and 65% in patient 4 on day28, coinciding with the marked reduction of leukemic blasts tomolecular negativity in both patients. This phenomenon was notsolely explained by the reduction ofmyeloid cells during therapy,but by an absolute expansion of lymphocytes. Indeed, a 10-foldincrease of absolute CD8þ lymphocyte numbers, a 38-foldincrease of NK cells, and a 24-fold increase of gd T cells frombaseline have been observed on day 28 in patient 4 (Fig. 4D).Comprising 45% to 50%of lymphocytes, NK cells represented thepredominant lymphocyte fraction in the bone marrow in bothpatients, with maximum absolute numbers of 2,798/mL and

4,933/mL. In microscopy of bone marrow aspirates, clusters ofcells consisting of lymphocytes tightly attached to leukemic blastssuggestive of immunological synapse formation have been fre-quently observed (Fig. 4E).

T cells with regulatory phenotype (CD4þ/CD25þ/FoxP3þ;Treg), evaluated in patient 4, accounted for approximately 3%of CD4þ T cells in peripheral blood and bone marrow beforetreatment and expanded during therapy to 20% in both com-partments on day 28.

DiscussionThe increased angiogenic activity in AML bone marrow has

provided the scientific rationale for the clinical evaluation of

Table 1. Summary of adverse events (highest grade observed according to the Common Terminology Criteria for Adverse Events, version 4.03)

Adverse event Grade Comments

Patient 1Fever 2 Fevers up to 39.0�Condays 1 to 3 after infusion of F16-IL2,manageablewith oral acetaminophen in the first three infusions

of F16-IL2Chills 1Fatigue 2Pain 2 Pain in lymph nodes/chloroma lesions after the first two infusions of F16-IL2GVHD 3 Significant worsening of GVHD occurred after the third infusion of F16-IL2, managedwith oral and topical glucocorticoidsSkin (stage) 3Liver (stage) 2

Cytokine-release syndrome 3 Hospitalization on day 25 after repeated infusions of F16-IL2 due to persisting high-grade fever, respiratory distress,exacerbation of skin GVHD (erythroderma), and suspected pulmonary infection; discharge on day 39

Fever 3 Temperature escalated to 40.5�C on day 25, resolved within 2 days of high-dose glucocorticoidsHypotension 2 Only fluids, no vasopressorsPleural effusion 2 Nonmalignant pleural effusion, left thoracocentesis necessaryDyspnea 3Weight gain 2 Weight increased by 5 kg, normalized at discharge

Pulmonary infiltrates 3 Middle lobe infiltrates compatible with atypical pneumoniaAnemia 2Thrombocytopenia 4 Prolonged thrombocytopenia after the first cycle, resolved to grade 3 on day 73 and to grade 2 on day 84Neutropenia 2

Patient 2Fever 2 Fever and chills persisted for 2 days after each infusion of F16-IL2 (peak temperature 39.5�C), manageable with

acetaminophenChills 1Fatigue 2GVHD 1 Signs of mild skin GVHD developed during the second cycle, resolved with topical steroidsSkin (stage) 1

Anemia 3Thrombocytopenia 4Neutropenia 4

Patient 3Cytokine-release syndrome 3 Signs of cytokine release and capillary leak syndrome developed in the first few days after treatment initiation;

hospitalization on day 1, discharge on day 15Fever 2 Peak temperature 39.7�Ca fewhours after thefirst infusionof F16-IL2; fevers persisted for 1week and resolved after 4 days

of glucocorticoidsChills 1Nausea 2Elevated creatinine 2 Creatinine levels transiently increased to 2.1 mg/dL, normalized within 4 daysWeight gain 2 Increase of body weight (8 kg) due to generalized edema, normalized by day 14Dyspnea 2

Anemia 3Thrombocytopenia 4Neutropenia 4

Patient 4Fever 2 Fever and chills typically occurred after the infusion of F16-IL2 (peak temperature 39.0�C), persisted for 2 to 3 days after

each infusion, and responded well to oral paracetamolChills 1Fatigue 2Anemia 2Thrombocytopenia 3 Normalized on day 22Neutropenia 4 Resolved to grade 3 on day 25 and to grade 2 on day 40

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antiangiogenic agents in AML (8, 13–15). However, therapeuticoutcomes of angiogenesis inhibitors in AML have been mostlydisappointing so far (16, 17), stimulating the search for alterna-tive strategies that take therapeutic advantage of angiogenic neo-vessels. Here, we report first-in-human experiences with thetargeted delivery of IL2 to the leukemia-associated vasculatureand stroma using the immunocytokine F16-IL2 in heavily pre-treated AML patients. We have chosen the situation after allo-HSCT to allow for a graft-versus-leukemia (GVL) response of atransplanted cellular immune system to the immunocytokine,and because we had previously treated 2 patients with late-stagerefractory AMLwithout previous allo-HSCT and hadnot observedsignificant antileukemic activity (data not shown).

The therapeutic principle of allo-HSCT is based on an activedonor immune system that fights tumor cells of the host. Wehypothesized that accumulating high doses of IL2 in the bonemarrow as the site of origin of the disease would promote GVLactivity in post–allo-HSCT AML patients. Indeed, immunocyto-kine treatment resulted in a dramatic expansion of cytotoxiceffectors, whichwas paralleled by a striking reduction of leukemicblasts or extramedullary AML lesions. It was unexpected that only

two infusions of the immunocytokine in combinationwith LDACwould result in a (transient) complete molecular clearance of theleukemic clone in 2 of 3 patients and in a complete metabolicresponse of extramedullary AML lesions as early as day 14 aftertreatment initiation. The antileukemic effects observed with thiscombination therapy are highly encouraging, given that allpatients were heavily pretreated with multiple lines of intensivetherapy, with 2 of the 4 patients having received even two allo-HSCTs. The observation that even patients who were previouslyrefractory to high-dose cytarabine (3 g/m2) responded supports asubstantial contribution of the targeted cytokine to the therapeu-tic effect. In fact, the cumulative dose of cytarabine (100 mg percycle)was 3 to4 times lower than thedoses that are usually used inLDAC regimens (10 mg/m2 or 20 mg twice daily for 10 days;ref. 18). Furthermore, LDAC alone has not been shown to induceremissions in patients with complex karyotype AML (18).

The targeted delivery of IL2 to the bonemarrow led to amassiveexpansion of immune effector cells, with CD8þ T cells and NKcells representing the most upregulated lymphocyte subsets.These observations are encouraging, in light of the fact that theyreproduce preclinical findings, which revealed a dominant role of

Figure 4.Immunologic effects of F16-IL2 therapy.A, changes in peripheral bloodlymphocyte subpopulations duringtherapy with F16-IL2 and LDAC asrevealed by flow-cytometric analysis.The most prominent increase wasobserved in CD16þ/CD56þ NK cells andCD8þ lymphocytes. Triangle arrowsindicate infusions of F16-IL2. B,photographs of May-Gr€unwald–Giemsa–stained peripheral blood smears showactivated lymphocytes and largegranular lymphocytes (obtained frompatient 2 after two infusions of F16-IL2).C, immunohistochemical staining ofpre- and posttherapeutic bone marrowbiopsies showedamassive influxofCD3þ

and CD8þ lymphocytes as exemplified inpatient 2 (CD3 staining) and patient 4(CD8 staining). D, flow-cytometricanalyses of bone marrow aspiratesshowed a 10-fold increase of CD8þ

lymphocytes anda38-fold increase ofNKcells at the end of the first cycle (patient4). E, cell clusters reminiscent ofimmunological synapse formationbetween activated lymphocytes andleukemic cells could be observedfrequently in the bone marrow. ALC,absolute lymphocyte count.






CD8þ and NK cells for the therapeutic activity of IL2-basedimmunocytokines in AML-bearing mice (5).

In line with the broad immunomodulatory properties of IL2,Tregs also increased during therapy. It is entirely conceivable thatTregsmight have negatively influenced the therapeutic GVL effect.Recent studies, however, which have used ultralow doses ofunconjugated IL2 to preferentially induce Tregs in an attempt toameliorate GVHD, did not reveal a diminished antileukemiaactivity or an enhanced risk of leukemia relapse after allo-HSCT(19, 20). Indeed, in the context of GVHD, Tregs were shownto allow for robust GVL activity in several leukemia models(21–23). Interestingly, there is evidence that Tregs do not impairthe immune response against tumors residing within the bonemarrow, whereas they readily suppress antitumor immunity ifthe same tumor is located outside the bonemarrow (24, 25). Thisphenomenon has been explained by an IL1b- and IL6-mediatedneutralization of the suppressive activity of Tregs by the bonemarrow stroma (in part through conversion of Tregs into IL17-producing T cells), leading to a conditional loss of Treg function inthe bone marrow and providing a favorable environment foreffector cells to mediate antitumor immunity (24). Thus, it is notentirely obvious that alternative immunostimulatory cytokinesthat do not significantly act on Tregs (e.g., IL15) would representbetter effector functions for bone marrow–targeted immunocy-tokines, especially in patients with relevant GVHD. In fact, it ispossible that the peripheral expansion of Tregs may have pre-vented even more severe manifestations of GVHD in patient 1while still allowing for significant antileukemic activity of cyto-toxic effectors at extramedullary sites. Combination strategies,however, which keep Treg levels low (e.g., anti–CTLA-4 antibo-dies; ref. 26), might be even more efficient, yet at the expense of apotentially higher toxicity.

The fact that we observed aggravation rather than ameliora-tion of GVHD symptoms contrasts with the notion of using IL2to stimulate Tregs and suppress GVHD (19, 20). Weekly IL2equivalents that were administered in our patients wereapproximately 10 to 100 times higher than the doses used forGVHD. The difference of the dose of IL2 available in the bonemarrow is likely to be even higher, when taking into accountthe targeting aspect. Indeed, although CD8þ T cells and gd Tcells did not change and NK cells only doubled under treatmentwith low-dose IL2 (19), we have observed an up to 38-foldexpansion of NK cells, a 10-fold expansion of CD8þ T cells, anda 24-fold increase of gd T cells in the bone marrow. Thus, thelocal dose of IL2 equivalents is likely to determine the netoutcome of IL2-induced stimulation of proinflammatory andsuppressive lymphocytes.

Although F16-IL2 was well tolerated in 2 patients, the other 2patients had substantial toxic side effects that either occurredimmediately after the first infusion or accumulated after severalinfusions of F16-IL2. The cytokine-release syndrome that devel-oped in both patients was manageable with high-dose glucocor-ticoids and did not reach the severity reported for patients receiv-ing chimeric antigen receptor–modified T cells (27). Both patientstolerated reexposure to lower doses of F16-IL2.

In principle, it is conceivable that the observed therapeuticeffects could have also partially been achieved with the admin-istration of free, unconjugated IL2. However, several lines ofevidence strongly support the contribution of the targeting aspectto the therapeutic efficacy. First, the most pronounced changes ineffector cell populations have been observed in the bone marrow

as compared with peripheral blood (e.g., up to 38-fold vs. 17-foldincrease inNK cell numbers in bonemarrow vs. peripheral blood,respectively), which we would not expect from a nontargetedapplication of IL2. The fact that we still have seen considerableimmunologic effects in the peripheral blood (and systemic toxi-cities) could be attributed to the "fluid" nature of the bonemarrow, which is—in contrast with solid tumors—part of theimmune system and dynamically connected to the peripheralblood, allowing immune effector cells to traffic between bonemarrow and the periphery and to aggravateGVHDeffects. Indeed,a comparable IL2-based immunocytokine only led to a modestincrease in peripheral effector cell counts in patients, in whichsolid tumors have been targeted (28). A dose-finding phase Istudy, which is currently in preparation, will show whether lowerstarting doses of IL2 equivalents may lead to a more prominentdiscrimination between bone marrow and peripheral bloodimmune effects. Second, the delivery of IL2 to the bone marrowwas demonstrated using immunohistochemical analyses of post-infusion bone marrow biopsies. Stromal structures decoratedwith IL2 were detectable 6 days after the last administration ofF16-IL2, indicating that the immunocytokine exhibited on-targetpersistence times of several days in vivo. In patients with solidtumors, comparable on-target persistence times have beendescribed for vascular-targeting antibodies (29–33). The lack ofposttherapeutic IL2 bone marrow signals in patient 4 might beexplained by interindividual differences in targeting performancein vivo. Interestingly, preliminary data suggest that angiogenesis-related matrix proteins may persist even after normalization ofmicrovessel density in patients achieving a CR (5), potentiallymaking TnC-A1 an attractive target for targeted cytokines also in apostremission setting. Third, patient 1 developed pain at extra-medullary AML sites after each of the first two infusions of theimmunocytokine, a side effect that is not commonly observedwith nontargeted IL2. Tumor pain upon infusion has also beenobserved in patients with solid tumors receiving L19-IL2, animmunocytokine that targets EDB-Fn (28, 34). Last, preclinicalexperiments using different syngeneic and xenograft models ofAML have clearly shown that complete leukemia eradicationscould only be achievedwith vascular-targeted IL2 in combinationwith cytarabine, whereas equimolar amounts of IL2 fused to anantibody of irrelevant specificity were ineffective in inhibitingleukemia progression (5).

In summary, a combination of F16-IL2 and LDAC can provideclinicallymeaningful benefit to patients in the desperate situationof AML relapse after allo-HSCT. We believe that F16-IL2 com-bined with either LDAC or with additional immunologic man-euvers to increase on-target activity of the stimulated effector cellsat the bonemarrow site deserves further evaluation in prospectiveclinical trials, with a dedicated part on dose finding, as it appearsthat patients with an allogeneic immune system may toleratelower doses of F16-IL2 as compared with patients with solidtumors.

Disclosure of Potential Conflicts of InterestD.Neri is a boardmember, has ownership interest (including patents), and is

a consultant/advisory board member for Philogen. No potential conflicts ofinterest were disclosed by the other authors.

Authors' ContributionsConception and design:C. Schliemann, K.L. Gutbrodt, T. Kessler, R.M.Mesters,Claudia Rossig, D. Neri, W.E. Berdel

Schliemann et al.





Development of methodology: C. Schliemann, S. Wiebe, R.M. Mesters,W.E. BerdelAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C. Schliemann, M. Pohlen, S. Wiebe, G. Silling,R.M. Mesters, B. Altvater, I. Gr€unewald, G. K€ohler, M. Stelljes, W.E. BerdelAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): C. Schliemann, T. Kessler, B. Altvater, C. Rossig,E. Wardelmann, W.E. BerdelWriting, review, and/or revision of themanuscript:C. Schliemann,M. Pohlen,S.Wiebe, T. Kessler, R.M.Mesters, L. Giovannoni,M. Sch€afers, C. Rossig, D.Neri,M. Stelljes, W.E. BerdelAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): C. Schliemann, G. Silling, L. Giovannoni,I. Gr€unewald, E. Wardelmann, D. Neri, W.E. BerdelStudy supervision: C. Schliemann, R.M. Mesters, W.E. Berdel

Other (involved in patient care and treatment): A. KerkhoffOther (patient care and treatment, immunohistochemical analyses ofTnC-A1 expression, and edited and approved the article): L. AngenendtOther (morphologic and immunohistochemical analysis): E. Wardelmann

AcknowledgmentsC. Schliemann, M. Sch€afers, and W.E. Berdel are supported by Deutsche

Forschungsgemeinschaft, DFG EXC 1003.The costs of publication of this articlewere defrayed inpart by the payment of

page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received September 23, 2014; revised January 5, 2015; accepted January 30,2015; published OnlineFirst February 11, 2015.

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Schliemann et al.




2015;3:547-556. Published OnlineFirst February 11, 2015.Cancer Immunol Res Christoph Schliemann, Katrin L. Gutbrodt, Andrea Kerkhoff, et al. Stem Cell TransplantationAcute Myeloid Leukemia Relapsing after Allogeneic Hematopoietic Targeting Interleukin-2 to the Bone Marrow Stroma for Therapy of

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