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Therapeutic Discovery

An Antibody Fusion Protein for Cancer ImmunotherapyMimicking IL-15 trans-Presentation at the Tumor Site

Vanessa Kermer, Volker Baum, Nora Hornig, Roland E. Kontermann, and Dafne M€uller

AbstractCytokines driving the immune response are powerful tools for cancer immunotherapy, but their

application is generally limited by severe systemic toxicity. Targeted approaches by means of antibody–

cytokine fusion proteins might enable focus on the cytokine activity to the tumor site, thereby reducing

unwanted side effects. Here, we investigated the possibility to improve the efficiency of interleukin (IL)-15

presentation in a targeted approach by the incorporation of an IL-15Ra chain fragment, mimicking

physiologic trans-presentation. Therefore, an antibody cytokine fusion protein (scFv_RD_IL-15) composed

of an antibody moiety targeting the tumor stromal fibroblast activation protein (FAP), an extended IL-

15Rasushi domain (RD) and IL-15 was generated, exhibiting antibody-mediated specific binding and

cytokine activity in soluble and targeted form. Comparative analysis with a corresponding antibody fusion

protein devoid of RD (scFv_IL-15) showed for scFv_RD_IL-15 in solution enhanced stimulatory activity on

Mo7e (IL-15Rbg) cells and reduced proliferation response on CTLL-2 (IL-15Rabg) cells, while in FAP-

targeted, that is, membrane-bound form, comparable proliferation of CTLL-2 (IL-15Rabg) cells was obtained.

In addition, scFv_RD_IL-15 achieved in its soluble and target-bound form stronger proliferation and

cytotoxicity on unstimulated and activated T cells, respectively. Furthermore, in vivo analysis in a lung

metastasis tumor mouse model revealed a superior antitumor effect for scFv_RD_IL-15 in comparison with

that obtained by an untargeted or RD missing version of IL-15 fusion protein. Thus, tumor-directed trans-

presentation of IL-15 in association with RD in form of an antibody fusion protein seems to be a promising

approach to further improve the antitumor effect of IL-15. Mol Cancer Ther; 11(6); 1279–88. �2012 AACR.

IntroductionThe application of cytokines to induce or promote the

generation of an effective antitumor immune response isan attractive approach in cancer immunotherapy. Amonga continuous growing list of cytokines with antitumorpotential (1), interleukin (IL)-15 appears as a candidatewith especially promising features (2, 3).IL-15 belongs to the 4 a-helix bundle cytokine family

andwas initially described in relation to IL-2, with whomit shares several biological functions, for example, theinduction of proliferation and differentiation of naturalkiller (NK), T, and B cells (4, 5, 6). Currently, the majorfunction of IL-15 appears to focus on the generation,proliferation, and activation of NK cells and the supportof the survival of CD8þmemory T cells (7, 8). Unlike IL-2,

IL-15 rather inhibits activation-induced cell death (9) andseems not to exert crucial influence on regulatory T cells(10), important properties in view of an immunothera-peutic application.

IL-15 and IL-2 have 2 receptor units (IL-2/15Rb and gc)of their heterotrimeric receptors in common, while thethird receptor unit (IL-2Ra and IL-15Ra) is specific foreach cytokine. Functional differences observed betweenIL-2 and IL-15 presumably ascribe mainly to the interac-tionwith their respective a receptor unit. Although IL-2 issecreted by activated T cells and binds in a soluble form toits high-affinity heterotrimeric receptor (IL-2Rabg) onactivated cells, IL-15 acts mainly in a membrane-boundform, presented in trans in association with its specific IL-15Ra subunit by monocytes and dendritic cells (antigen-presenting cells, APC) to NK or CD8þ T cells, expressingthe heterodimeric intermediate affinity receptor IL-2/15Rbg (ref. 11; Fig. 1A). For the process of trans-presen-tation, intracellular IL-15 binds to the IL-15Ra chain thatshuttles to the cell surface where it presents IL-15 toneighboring cells expressing the receptor IL-2/15Rbg .Thus, a more directed and controlled action of the cyto-kine is achieved (12). The sushi domain of the IL-15Rachain has been identified as the structural region involvedin IL-15binding (13). There is also thepossibility that IL-15binds to soluble versions of IL-15Ra forming solublecomplexes with stimulatory or inhibitory activity. In this

Authors' Affiliation: Institut f€ur Zellbiologie und Immunologie, Universit€atStuttgart, Stuttgart, Germany

Note: Supplementary material for this article is available at MolecularCancer Therapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Dafne M€uller, Institut f€ur Zellbiologie und Immu-nologie, Universit€at Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.Phone: 49-711-685-66999; Fax: 49-711-685-67484; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-12-0019

�2012 American Association for Cancer Research.

MolecularCancer

Therapeutics

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on June 18, 2020. © 2012 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

Published OnlineFirst April 6, 2012; DOI: 10.1158/1535-7163.MCT-12-0019

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Figure 1. Schematic illustration of IL-15 trans-presentation (A) and the assembly of the fusion proteins (B). scFv, FAP-specific antibody fragment; VH,variable region of the heavy chain; VL, variable region of the light chain; RD, IL15Ra(sushi domain þ 12 amino acids of exon 3); L1, G4SG4SG2SA; L2,SG4SG4; L3, G3SG4SG3SG4SLQ; L4, SGSG4SLQ; black square, hexahistidyl-tag. Characterization of the fusion proteins by 10% SDS-PAGEunder reducing conditions (Coomassie staining; C) and Western blot (D). Recombinant protein (2 mg per lane) was detected via hexahistidyl-tag.E, antibody-mediated binding properties analyzed by flow cytometry. Binding specificity was determined by incubating the fusion proteins(350 nmol/L) on FAP-positive (B16-FAP) and FAP-negative (B16) cells, respectively. F, binding affinity was compared by titrating the fusion proteinson B16-FAP cells. Bound constructs were detected via the hexahistidyl-tag. Gray filled, cells; dotted line, detection system; black line, antibodyconstruct. Graphic shows mean � SD, n ¼ 3.

Kermer et al.

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case, soluble IL-15Ra might arise from proteolytic cleav-age of the membrane form by metalloproteinases (14, 15).The antitumor potential of IL-15wasobserved inmouse

models employing recombinant IL-15 (16, 17), antibody–IL-15 fusion protein (18), IL-15 gene-modified tumor cells(19, 20), and IL-15 transgenic mice (21). The relevance ofIL-15 trans-presentation for an efficient antitumorresponse was shown in a mouse model with IL-15Ra–transfected MC38 tumor cells. Here, the expression of IL-15Ra dose dependently delayed the formation of fatalpulmonary metastases through the activation of NK cells(22). Subsequently, it was shown in vitro and in vivo thatsoluble complexes of recombinant IL-15 and IL-15Raweremuch more stimulatory than soluble IL-15 alone (23).Treatment of mice with IL-15xIL15RaFc complexesenhanced the proliferation of CD8þ memory T cells andNK cells in vivo. Furthermore, tumor burden could bereduced and survival extended in a systemic B16 mela-nomamousemodel (24, 25). Similar results were obtainedby an IL-15/IL-15Ra domain fusion protein (26, 27).We hypothesize that targeting a fusion protein consist-

ing of IL-15 and an extended IL-15Rasushi domain by anantibody moiety to the tumor site will mimic locally thetrans-presentation of IL-15 by the IL-15Ra, enhancing itsstimulatory potential, thus further improving the antitu-mor effect of IL-15 (Fig. 1A). In this study, we report thegeneration of the antibody–cytokine fusion proteinscFv_RD_IL-15, composed of the fibroblast activationprotein (FAP)-specific scFv (scFv36), an IL-15Ra fragment(RD) and IL-15.We show antibody-mediated binding andcytokine activity for this construct in solution or targetedform and describe the influence of the RD on the receptoractivation properties of the fusion protein. Proliferation ofan IL-15Rbg receptor-expressing cell line (Mo7e) wasenhancedby theRD-containing fusionprotein in solution,whereas proliferation of a cell line expressing the IL-15Rabg receptor (CTLL-2) was reduced. Nevertheless, intarget-bound form, the proliferation effect on CTLL-2was mainly RD independent. Also, peripheral bloodmononuclear cells (PBMC) could be effectively stimulatedby scFv_RD_IL-15 in targeted and soluble form. Here,scFv_RD_IL-15 was more efficient than scFv_IL-15enhancing the proliferation and cytotoxicity response ofunstimulated and activated T cells, respectively. Further-more, scFv_RD_IL-15 was more effective than scFv_IL-15or RD_IL-15 in the treatment of a lung metastasis mousemodel. Thus, the combination of antibody-mediatedtumor targeting and IL-15Ra domain associated trans-presentation of IL-15 in formof a fusionprotein appears asa promising approach for cancer immunotherapy.

Materials and MethodsMaterialsAntibodies were purchased from Biolegend (aCD3-

PerCP and aCD8-PE), Immunotools (aCD4-PE andaCD56-PE), Miltenyi Biotec (ahexahistidyl-tag-PE),and Santa Cruz (aIL-15, ahexahistidyl-tag-HRP, and

aCD107a-FITC). Recombinant human IL-2 and granulo-cyte-macrophage colony-stimulating factor (GM-CSF)were obtained from Immunotools and recombinantFAP from R&D Systems. B16-FAP and B16 cells (K. Pfi-zenmaier, IZI) were cultured in RPMI-1640, 5% FBS.Mo7e (H. van der Kruip, IKP) and CTLL-2 (P. Scheurich,IZI) were cultured in RPMI-1640, supplemented with20% FBS, 10 mmol/L HEPES, 2 mmol/L L-glutamine,0,13 mmol/L L-asparagine, 0,05 mmol/L b-mercapto-ethanol, 1 mmol/L natriumpyruvate, nonessential aminoacids, and 10 ng/mL rhGM-CSF (Mo7e) or 400 IU/mLrhIL-2 (CTLL-2), respectively. Cells were tested for myco-plasms and their morphologic appearance monitored bymicroscopic means. Antigen (FAP) expression and cyto-kine growth dependence of the respective cell lines wasverified by flow cytometry and proliferation assays,respectively. PBMCs were isolated from buffy coat ofhealthy donors (blood bank, University Medical CenterUlm) byFicoll gradient andwere cultivated inRPMI-1640,10% FBS. C57BL/6JRj mice were purchased from ElevageJanvier. Animal care and all experiments carried out werein accordance with federal guidelines and have beenapproved by university and state authorities.

Generation of the recombinant fusion proteinsThe procedure is described in the Supplementary Data

S1.

Binding analysisA total of 2.5� 105 cells (B16-FAP/B16) were incubated

with fusion protein (200 nmol/L or titrated, accordingly)for 2 hours at 4�C. After washing, cells were incubatedwith a hexahistidyl-tag directed phycoerythrin (PE)-con-jugated mouse monoclonal antibody, for 1 hour at 4�C.Washing and incubation stepswere carried out in PBS, 2%FBS, and 0.02% sodium azide. Finally, cells were analyzedin a Cytomics FC500 (Beckman Coulter).

Proliferation assays with cell linesCytokine growth-dependent cells (CTLL-2/Mo7e)

were washed and suspended in culture medium withneither IL-2 nor GM-CSF. A total of 20,000 cells per wellwere seeded in 96-well plates and starved for 4 hours.Then the fusion proteins were added to the cells. After 3(CTLL-2) or 4 (Mo7e) days, proliferation of the cells wasmeasured by MTT assay (28). The cytokine activity of thefusion proteins presented in an antibody-mediated mem-brane-bound form was analyzed in the following setting:10,000 cells per well (B16-FAP/B16) were seeded in a 96-well tissue culture plate. Next day, cell growth wasarrested by treatment with 10 mg/mL mitomycin C (Sig-ma), for 2 hours at 37�C. Cells were washed twice withmedium before addition of the fusion proteins. After 1hour incubation at room temperature, the supernatantwas discarded and adherent cells washed twice withmedium. Then previously starved CTLL-2 cells (20,000cells per well) were added and incubated for 3 days.Finally, the cells in suspension (CTLL-2) were carefully

Mimicking IL-15 trans-Presentation for Cancer Immunotherapy

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transferred to a new 96-well plate, followed by the MTTassay (28).

Proliferation assays with PBMCsPBMCs were stained at a concentration of 1 � 106

cells/mL with 625 nmol/L carboxyfluorescein diacetatesuccinimidyl ester (CFSE) using the CellTrace CFSECell Proliferation Kit (Life Technologies), following theinstructions of themanufacturer. Proliferation induced bythe fusion proteins in solutionwas assessed by incubating2 � 105 PBMCs per well with the corresponding fusionprotein for 5 days, followed by flow cytometry analysis.Additional antibody-mediated staining was carried outto identifyT cells (CD3-PerCP), CD4þTcells (CD3-PerCP/CD4-PE), CD8þT cells (CD3-PerCP/CD8-PE), and NKcells (CD3-PerCP/CD56-PE).

To determine PBMC proliferation induced by targetedfusion proteins, recombinant FAP (3 mg/mL) was coatedovernight at 4�C on an ELISA plate. Fusion proteins (1mmol/L) were added and incubated for 1 hour at roomtemperature. After washing with PBS, 2� 105 PBMCs perwell were added. Alternatively, the fusion proteins (50,100, 200 nmol/L) were incubated for 1 hour with 2 � 105

B16-FAP cells per well. Then, B16-FAP cells were washedand 2 � 105 preactivated PBMCs per well added. After 5days, proliferation of PBMCs or T cells (additional CD3-PerCP staining) was determined by flow cytometry. Pre-activated T cells were used in the targeted assay, ascoculturewith B16-FAP cells inhibited the responsivenessof unstimulated T cells. Preactivation of PBMCs wasachieved by coculture with adherent B16-FAP cells inpresence of 2.5 pmol/L bispecific antibody scDbFAPCD3for 24 hours. Subsequently, PBMCs were harvested andwashed before their addition to the assay as indicatedabove.

Cytotoxicity assayCytotoxic potential of T cells was assessed in terms of

degranulation and determined by measuring CD107aexposure on the cell surface by flow cytometry analysis.In the first step, 2 � 105 unstimulated PBMCs per wellwere incubated with the fusion proteins in solution oralternatively 2 � 105 preactivated PBMCs (as indicatedabove)were incubatedwith the fusionproteins targeted toB16-FAPcells (as indicated above).After 5days, cellswerewashed and cytotoxic T-cell degranulation triggered. Atotal of 2� 104 B16-FAP cells perwellwere seeded the daybefore and incubated with 30 pmol/L bispecific antibodyscDbFAPCD3 for 1 hour, before the addition of pretreatedPBMCs for additional 6 hours in presence of 1.4 mL perwell BD GolgiStop (monensin; BD Biosciences). Degran-ulation of T cells was monitored by CD107a-FITC/CD3-PerCP staining and flow cytometry analysis.

PharmacokineticsGroups of 5 C57BL/6JRj mice (4–6 months) were

injected i.v. with 1 nmol fusion protein per mouse. Bloodsamples were taken at different time intervals (3, 30, 60

minutes, 2, 6, and 24 hours) from the tail and serumconcentration of the fusion proteins analyzed bysandwichELISA. Inbrief, anti–IL-15monoclonal antibody(3 mg/mL)was coated.After blocking, serumandpurifiedfusion proteins were titrated and bound fusion proteindetected via horseradish peroxide (HRP)-conjugatedmouse hexahistidyl-tag–specific antibody as describedelsewhere (29). Serumconcentration of the fusionproteinswas determined by interpolating the corresponding stan-dardcurve. For comparison, thefirstvalue (3minutes)wasset to 100%. Pharmacokinetic parameters [(t1/2a, t1/2b,area under the curve (AUC)] were calculated via Excelusing the first 3 to 4 time points to calculate t1/2a and thelast 3 measurable time points to calculate t1/2b. AUC wasdetermined for the time interval of the first 6 hours.

B16-FAP lung metastasis modelC57BL/6JRj mice (4 months) were injected i.v. with 8.5

� 105 B16-FAP cells per animal. Groups of 5 to 6 animalswere treated with PBS, scFv_IL-15, scFv_RD_IL-15, orRD_IL-15, respectively. Treatment of 0.2 nmol fusionprotein was applied i.p. on days 1, 2, and 10 after cellinjection. Mice were sacrificed on day 20. Lungs wereremoved and metastasis counted.

Statistical analysisFor comparison ofmultiple groups in the in vitro experi-

ments, the one-way ANOVA followed by the Tukey posttest was applied. Statistical analysis of the in vivo experi-ments was conducted by Student t test (GraphPad Prismsoftware).

ResultsGeneration of the fusion proteins

The fusion protein scFv_RD_IL15 is composed of anantibody moiety, a receptor domain, and a cytokine com-ponent. The antibody component corresponds to an scFvthat consists of the variable regions of the heavy and lightchain of the antibody mo36 (ref. 30; scFv36) directedagainst the tumor stromal FAP. This antibody is cross-reactive in mice and humans and had been previouslygenerated at the institute (30). It was used in our study totarget a stably FAP-transfected B16 melanoma cell line,suitable for in vitro and in vivo studies. Thus, FAP is usedhere as a model antigen to evaluate the feasibility of ourapproach, which might be extended later on to othertumor targets. The receptor domain (RD) comprises theamino acids 31 to 107 of thehuman IL-15Ra (sushi domainplus the subsequent 12 amino acids). The cytokine com-ponent corresponds to human IL-15. In the scFv_RD_IL-15 construct, the scFv36 is located at the N-terminus,followed by the RD and the IL-15 domain. Furthermore,scFv_IL-15, an antibody fusion protein without RD wasgenerated as well as the control fusion proteins scFv_RDand RD_IL-15, missing IL-15 and scFv, respectively.All constructs are provided with a hexahistidyl-tag at theC-terminus (Fig. 1B).

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The fusionproteinswereproduced in stably transfectedHEK293 cells and purified by immobilized metal ionaffinity chromatography with yields of 2.6 mg/L(scFv_RD_IL-15), 0.26 mg/L (scFv_IL-15), 0.66 mg/L(scFv_RD) and 0.88mg/L (RD_IL-15), respectively. Anal-ysis of the purified proteins by SDS-PAGE (Coomassiestaining) under reducing conditions (Fig. 1C) revealedhigh degree of purity and a single band for the scFv_RDconstruct (36 kDa), pronounced double bands for thefusion proteins scFv_RD_IL-15 (48 and 53 kDa) andRD_IL-15 (24 and 28 kDa), and 3 bands for scFv_IL-15(42, 46, and 50 kDa). The IL-15 sequence comprises 3potential N-linked glycosylation sites. The N-glycosy-lated nature of the fusion proteins with IL-15 componentwas shown by PNGase F digestion of the proteins, result-ing in a reduction of the slower migrating bands in SDS-PAGE (data not shown). The bands correlated with thepredicted molecular mass of the fusion proteins:scFv_RD_IL15 (51.4 kDa), scFv_IL-15 (41.5 kDa), scFv_RD(37.2 kDa), and RD_IL-15 (25 kDa). The identity of thefusion proteins was further confirmed by immunoblotanalysis with an anti–hexahistidyl-tag antibody (Fig. 1D).High-performance liquid chromatography analysis ofscFv_RD_IL-15 and scFv_IL-15 revealed a mainly homo-geneous population, taking into account the glycosylationstatus of these proteins. A second minor peak probablycorresponds to a dimeric form, as scFvsmight dimerize toa certain degree. No higher aggregate formation wasobserved (Supplementary Data S2).

Binding propertiesSpecific binding of the antibody fusion proteins to FAP

was shown by flow cytometry (Fig. 1E). Stably FAP-

transfectedB16 cells (B16-FAP)wereused as FAP-positivecell line, and B16 wild-type cells (B16) were used as FAP-negative control cell line. ScFv_RD_IL-15, scFv_RD, andscFv_IL-15 showedbinding toFAP-positive B16-FAPcellsbut not to FAP-negative B16 cells. No binding wasdetected for RD_IL-15 neither on B16-FAP nor on B16cells. Furthermore, titration of the antibody fusion pro-teins revealed similar binding affinity of scFv_IL-15 andscFv_RD_IL15 that was approximately 7- and 5-foldhigher than the binding affinity shown by the scFv andscFv_RD, respectively (Fig. 1F).

Cytokine activity of the fusion proteinsThe cytokine activity of the fusion proteins was ana-

lyzed in proliferation assays with the cytokine growth-dependent cell lines CTLL-2 and Mo7e. Human IL-15induced the proliferation of both cell lines, allowing theanalysis of stimulatory properties in the context of differ-ent IL-15 receptor chain compositions. CTLL-2, a murinecytotoxic T lymphocytic cell line, expresses IL-15Ra andIL-15Rbg , whileMo7e, a humanmegakaryocytic leukemiccell line, expresses IL-15Rbg chains only. Proliferationassays with the fusion proteins in solution revealed cyto-kine activity on both cell lines for all fusion proteinscontaining IL-15 (Fig. 2A and B). On CTLL-2 cells,scFv_IL-15 showed high activity at picomolar concentra-tions, while fusion proteins comprising the RD fragment(scFv_RD_IL-15 and RD_IL-15) were clearly less active(approximately 50-fold, comparing constructs with andwithout scFv, respectively; Fig. 2A). In contrast, on Mo7ecells, proliferation in response to scFv_RD_IL-15 andRD_IL-15 was also comparable but significantly moreactive (20- to 30-fold) than scFv_IL-15 (Fig. 2B). The scFv

Figure 2. Cytokine-dependentproliferation assays with the fusionproteins in solution were carried outwith CTLL-2 (IL-15Rabg ; A) andMo7e (IL-15Rbg ; B) cells. Cytokineactivity of target-bound fusionproteinswas shownby incubation onB16-FAP (C) or B16 (D) cells,followed bywashing and the additionof CTLL-2 cells. Indicated areconcentrations of the constructsduring the incubation step on targetcells. After 3 (CTLL-2) or 4 (Mo7e)days, proliferation was measured byMTT assay. Graphics show mean �SD, n ¼ 3.






and the scFv_RD did not show any stimulatory effectneither on CTLL-2 nor on Mo7e cells.

Next, the cytokine activity of the constructs was inves-tigated in their membrane-bound form, targeting the FAPantigen on the cell surface, thereby mimicking a trans-presentation of IL-15 either in presence (scFv_RD_IL-15)or in absence (scFv_IL-15) of the receptor a-chain domain(RD). The fusion proteins were incubated on B16-FAP orB16 cells and the IL-15 activity of bound constructs wasdetermined by the proliferation of cocultured CTLL-2cells. Here, proliferation was observed in response to theantigen-dependent membrane-bound presentation ofboth, scFv_RD_IL-15 and scFv_IL-15 (Fig. 2C). Unlike insolution, both constructs displayed a similar proliferationeffect. As expected, no stimulation of CTLL-2 wasobserved for the experimental setting with FAP-negativeB16 cells (Fig. 2D).

Then, we analyzed the proliferation effect of the fusionproteins on PBMCs. Similar to the proliferation patternobserved for Mo7e cells, fusion proteins in solution pre-senting IL-15 and RD (RD_IL-15 and scFv_RD_IL-15)achieved a stronger proliferation response (11- to 15-fold)than the fusion protein with IL-15 only (scFv_IL-15). Alsoin this case, scFv_RD_IL-15 and RD_IL-15 were equallyeffective,while scFv_RDdidnot induceproliferation at all(Fig. 3A). In addition, scFv_RD_IL-15 and scFv_IL-15were also shown to be active in their target-bound form(Fig. 3B). Furthermore, we determined the proliferation

effect of scFv_RD_IL-15 and scFv_IL-15 in solution inregard to different immune cell populations (Fig. 3C).Here, NK and CD8þ T cells were more responsive toIL-15 stimulation than CD4þ T cells. Irrespective of thecell type, scFv_RD_IL-15 was significantly more effectivethan scFv_IL-15 at low concentration (1 nmol/L).Improved proliferation of T cells in response to IL-15presented in combination with RD was shown for thefusion proteins in solution (Fig. 4A) as well as for thefusion proteins in target-bound state (Fig. 4B). Forthe latter, scFv_IL-15 and scFv_RD_IL-15 were bound viathe antibody moiety to FAP-expressing cells (B16-FAP),presenting IL-15 or RD_IL-15, respectively on the cellsurface. Thus, by mimicking IL-15 trans-presentation, theproliferation effect on preactivated T cells was deter-mined, indicating an advantage for the membrane pre-sentation of IL-15 in an additional RD-bound form. Fur-thermore, the influence of the fusion proteins on thecytotoxic potential of T cells was analyzed (Fig. 4C andD). In solution, scFv_RD_IL-15 as well as scFv_IL-15 wasable to enhance the percentage of degranulating T cells,whereby at low concentrations (0.1 and 1 nmol/L)scFv_RD_IL-15 showed a stronger effect than scFv_IL-15 (Fig. 4C). Also in the target-bound form, scFv_RD_IL-15 was at lower concentration more effective thanscFv_IL-15, increasing the cytotoxic potential of preacti-vated T cells (Fig. 4D). Therefore, the fusion of RD to IL-15,presented either in soluble or targeted form, improves

Figure 3. Cytokine activity of fusionproteins induces PBMCproliferation. CFSE-labeledPBMCs were incubated either withfusion proteins in solution (A) orfusion proteins bound torecombinant FAP coated on ELISAplate (B). After 5 days, proliferationof PBMCs was measured by flowcytometry. C, PBMCs wereincubated with fusion proteins insolution as indicated above andproliferation of CD4þ T cells (CD3-PerCP/CD4-PE), CD8þ T cells(CD3-PerCP/CD8-PE), and NKcells (CD3-PerCP/CD56-PE)determined by appropriateantibody staining, followed by flowcytometry analysis. Graphics showmean � SD, n ¼ 3–4; ��, P < 0.01;��, P < 0.001.

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significantly the response of T cells in terms of prolifer-ation and cytotoxicity.

Pharmacokinetics and therapeutic potential in atumor mouse modelPharmacokinetic analysis in mice revealed some differ-

ences in thehalf-life of the fusionproteins. ScFv_RD_IL-15showed an approximately 2-fold longer t1/2a than RD_IL-15 and scFv_IL-15 (P < 0.05). t1/2b was similar forscFv_RD_IL-15 and RD_IL-15, but 6-fold (P < 0.01) and3.5-fold (P < 0.001) increased in comparisonwith scFv_IL-15. The AUC calculated for scFv_RD_IL-15 was approx-imately 1.7- and 2.5-fold higher than theAUCdeterminedfor RD_IL-15 and scFv_IL-15, respectively (P < 0.01; Table1; Fig. 5A).Finally, the therapeutic potential of the fusion proteins

was investigated in a B16-FAP lung metastasis mousemodel, whereat dosing and treatment schedule was cho-

sen based on data of the IL-15/IL-15Ra domain fusionprotein (RLI) in the literature (26). B16-FAP cells wereinjected i.v. into C57BL/6JRj mice on day 0 and treated ondays 1, 2, and 10 with a single dose of 0.2 nmol fusionprotein (scFv_RD_IL-15, scFv_IL-15, and RD_IL-15),respectively. After 20 days, lungs were removed and thenumber of tumor cell foci counted (Fig. 5B). In comparisonwith the PBS control group, all groups treated with IL-15fusion proteins showed significantly less metastasis.Strongest effect was achieved by treatment withscFv_RD_IL-15 (approximately 86% metastasis reduc-tion), followed by application of RD_IL-15 and scFv_IL-15 with similar outcome (approximately 46% and 53%metastasis reduction, respectively). Thus, comparing thetargeted versions of the IL-15 fusion proteins (scFv_IL-15and scFv_RD_IL-15), the presence of RD significantlyenhanced the therapeutic potential. On the other hand,comparing the targeted and nontargeted versions of RD

Figure 4. Effect of scFv_IL-15 andscFv_RD_IL-15 in soluble andtargeted form on T-cell proliferationand cytotoxicity. PBMCs wereincubated with the fusion proteinseither in solution (A andC) or targetedtoB16-FAP cells (B andD) for 5 days.Then, proliferation (A and B) wasdetermined for T cells identified bymonoclonal antibody (mAb) (CD3-PerCP) staining by CFSEmeasurement in flow cytometryanalysis. Alternatively, cytotoxicity ofT cells (C and D) was assessed byincubating PBMCs with B16-FAPcells in presence of 30 pmol/LscDbFAPCD3 and monensin for 6hours. Degranulating T cells wereidentified by mAb (CD3-PerCP/CD107a-FITC) labeling via flowcytometry. Unstimulated orpreactivated PBMCs were used forthe approach with fusion proteins insoluble and targeted form,respectively. Data of assays withtargeted fusion proteins arepresented as relative to the effect ofprestimulated PBMCs in absence offusion protein. Graphics show mean�SD,n¼3–6. �,P<0.05; ��,P<0.01;��, P < 0.001.

Table 1. Pharmacokinetic parameters of IL-15 fusion proteins

Fusion protein Molecular mass (kDA) t1/2a (min) t1/2b (h) AUC(0–6 h) (h�%)

scFv_RD_IL-15 51 58 � 18 8.5 � 4.0 190 � 29RD_IL-15 25 31 � 3 4.9 � 1.2 111 � 31scFv_IL-15 41 25 � 10 1.4 � 0.3 76 � 37






bearing IL-15 fusion proteins (scFv_RD_IL-15 and RD_IL-15) showed that the targeted form was more effective.Thus, scFv_RD_IL-15, combining tumor targeting and IL-15 presentation in the RD context, revealed the besttherapeutic potential. No signs of toxicity (e.g., weightloss) were observed during the therapeutic experiment.

DiscussionInitially, soluble IL-15Ra, generated by shedding from

human cell lines was observed to exert an antagonisticeffect. Binding of soluble IL-15Ra to soluble IL-15 led tocompetition with cell surface receptors inhibiting IL-15–mediated function (14). Later, complex formation ofrecombinant soluble IL-15 and IL-15Ra-Fc molecules wasreported to enhance the bioactivity of IL-15, inducingproliferation of NK cells and memory T cells in vitro andin vivo (23, 25). Structural studies identified the "sushi"region in the IL-15Ra as the main responsible element forIL-15 binding (31). In vitro, soluble complexes of recom-binant IL-15 and the IL-15Ra sushi domain weredescribed to induce agonistic effects on the intermediateaffinity receptor (IL-15Rbg), inducing stronger prolifera-tion than IL-15 alone (32). This effect could be even

enhanced when the sushi domain of IL-15Rawas extend-ed by 13 amino acids of the exon 3 (IL-15Rasushiþ),thereby increasing the binding affinity for IL-15 (33).Although this complex showed reduced bioactivity onhigh-affinity receptor IL-15Rabg–expressing cell lines, thecombination of IL-15 and IL-15Rasushiþ into a fusionprotein (RLI) retrieved amolecule that presentedmaximalenhancement of IL-15Rbg stimulation without losingstimulatory capacity for IL-15Rabg (32, 33). Furthermore,the fusion protein (RLI) showed efficiency in vivo byinducing antitumor effects in diverse mouse models(26). Therefore, for the generation of the targetedscFv36_RD_IL-15 fusion protein, we decided to relay onthe RLI model with the IL-15Rasushiþ variant buildingthe IL-15Ra domain (RD). We could show that the fusionto the scFv component did not interfere with the cytokineactivity of IL-15 in combination with the RD fragment, asour untargeted control construct RD_IL-15 displayedsimilar activity than scFv_RD_IL-15 in solution. On theother hand, fusion of the scFv to the cytokine componentand or the receptor unit did not impede specific binding ofthe construct to the tumor target. Thus, scFv_RD_IL-15and scFv_IL-15 boundwith similar affinity to FAP-expres-sing cells. Furthermore, we analyzed the cytokine activityof the antibody fusion proteins in the context of interme-diate (IL-15Rbg) and high-affinity receptor IL-15Rabg–expressing cell lines. In solution, constructs presentingIL-15 in combination with the IL-15Ra domain (RD)stimulated more efficiently the proliferation of IL-15Rbg–expressing Mo7e cells. This observation was inaccordance with the data reported for the (RLI) fusionprotein and the IL-15/IL-15Rasushiþ complex, showingenhanced cytokine effect on Mo7e cells in comparisonwith IL-15 alone (32, 33). In regard to the stimulation of thehigh-affinity receptor IL-15Rabg , the presence of the RDfragment in the fusion proteins decreased the cytokineactivity of the constructs in solution, independently of thepresence of the scFv. Thus, proliferation of CTLL-2 (IL-15Rabg) cells was inducedwith picomolar concentrationsof scFv_IL-15, but rather nanomolar concentrations ofscFv_RD_IL-15 and RD_IL-15. This resembles the resultsdescribed for soluble complexes of IL-15 and IL-15Rasushiþ on high-affinity receptor–expressing cells, inwhich the extension of the sushi domain interferes withIL-15 receptor stimulation, but differs from the stimula-tory effect described for the RLI fusion protein thatshowed to be equivalent or even better than IL-15 in termsof IL-15Rabg activation (33, 26). The lattermight be due tothe particular cell system used (Kit225 expressing inter-mediate and high-affinity receptors, in which intermedi-ate receptor activation is specifically blocked by an anti-body), although differences in the sterical presentationinherent to the configuration of the particular recombi-nant fusion protein cannot be excluded. Nevertheless, theinhibitory effect in cytokine activity observed forscFv_RD_IL-15 in solution was abolished in the targetedform, that is, bound toB16-FAPcells, presentingRD_IL-15in amembrane-bound form. Here, scFv_RD_IL-15 as well

Figure 5. A, pharmacokinetics: fusion proteins (1 nmol) were injected i.v.into C57BL/6 mice (n ¼ 4–5) and serum concentration determined atdifferent time points. B, therapeutic experiment: 8.5� 105 B16-FAP cellswere injected i.v. into C57BL/6 mice on day 0. Mice were treated with asingledose (0.2 nmol) fusionprotein (i.p.) ondays 1, 2, and10. Lungswereremoved on day 20 and tumor foci counted. Graphics show mean� SD,n ¼ 5–6. �, P < 0.05; ��, P < 0.01; ��, P < 0.001.

Kermer et al.





as scFv_IL-15 showed equivalent stimulatory potential onCTLL-2 cells. Thus, for targeted constructs, effective trans-presentation of IL-15 to IL-15Rabg–expressing cellsappeared independent of the association to RD.Resting NK and naive T cells express the intermediate

affinity IL-15Rbg phenotype (10, 22, 34). Thus, prolifera-tion assays with unstimulated PBMCs retrieved similarresults than those obtained with the Mo7e (IL-15Rbg) cellline in response to the fusion proteins in solution, achiev-ing stronger effects with scFv_RD_IL-15 than withscFv_IL-15. NK as well as T cells (CD8þ T cells and CD4þ

T cells) were identified to be responsive, following thisactivation pattern. Overall higher proliferation responseof CD8þ T cells and NK cells in comparison with CD4þ Tcells was observed, correlatingwith the report of very lowIL-15Rb expression levels on na€�ve CD4þ T cells (35). Inaddition, better performance of scFv_RD_IL-15 in com-parison with scFv_IL-15 was also determined for theconstructs in targeted form, that is, bound to FAP-expres-sing tumor cells, enhancing proliferation and cytotoxicityof activated T cells. Considering that for effector T cellshigh expression levels of IL-15Ra and therefore potentialexpression of high and intermediate affinity IL-15 recep-tors have been described (10), our results likely reflect thetarget-directed trans-presentation of RD_IL-15 to bothreceptor types. Even in this case, better performance ofscFv_RD_IL-15 in comparison with scFv_IL-15 appearsreasonable, considering that in regard to IL-15Rabg acti-vation, both constructs showed in targeted form compa-rable cytokine activity on CTLL-2 cells (IL-15Rabg), whilestimulation of IL-15Rbg is clearly favored by the presenceof RD in the construct.In vivo analysis of scFv_RD_IL-15, scFv_IL-15, and

RD_IL-15 in the B16-FAP lung metastasis mouse modelresulted in clear antitumor effects for all 3 constructs,corroborating the antitumor potential of this cytokine.The best result was achieved with scFv_RD_IL-15, fol-lowed by scFv_IL-15 and RD_IL-15 to similar extent.Pharmacokinetic studies in mice indicated small differ-ences between the constructs in half-life and AUC, favor-ing scFv_RD_IL-15.According to the in vitrodata obtainedwith these constructs, cytokine activity could be remark-ably enhanced by presenting IL-15 in association with thereceptor a domain to IL-15Rbg–expressing cells. Thiseffect was also reported for the RLI fusion protein incomparison with recombinant IL-15 (approximately fac-tor 500; ref. 26). Also here, in vivo analysis in a B16F10/lung metastasis model revealed a stronger antitumor

effect for RLI than for IL-15. Nevertheless, pharmacoki-netic analysis of RLI and IL-15 indicated a 6-fold and 34-fold increase in half-life and AUC for RLI, respectively,suggesting that improved pharmacokinetic properties aswell as enhanced cytokine activitymight contribute to thestronger antitumor effect observed for RLI. In our study,scFv_IL-15 and RD_IL-15 achieved similar antitumoreffects; an outcome probably influenced by the tumortargeting properties of scFv_IL-15. Enhanced antitumoractivity by directing IL-15 to the tumor in form of anantibody–cytokine fusion protein has been described pre-viously (18). Here, IL-15 was fused to a fibronectin extradomain-B (EDB)-specific antibody in a diabody format,resulting in ahomodimericmolecule.Accumulation at thetumor site as well as target-mediated tumor growth inhi-bition was shown in an s.c. F9 tumor mouse model,ascribing a crucial role for CD8þ T cells in the therapeuticeffect. Tumor directed targeting of IL-15/IL-15Ra as acomplex or fusion protein has not been reported so far.Here, bymeans of scFv_RD_IL-15,we could show that thestrategy of combining tumor targeting by a recombinantantibody and trans-presentation of IL-15 in the IL-15Radomain context was able to further enhance the antitumorpotential of this powerful cytokine. Thus, scFv_RD_IL-15appears as a promising reagent that sets itself apart fromother recombinant molecules developed for the applica-tion of IL-15 in cancer immunotherapy.

Disclosure of Potential Conflicts of InterestR.E. Kontermann is a consultant to BioNTech. No potential con-

flicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: D. MullerDevelopment of methodology: V. Kermer, V. Baum, N. HornigAcquisition of data: V. Kermer, V. BaumAnalysis and interpretation of data: V. Kermer, V. Baum, D. MullerWriting, review, and/or revision of the manuscript: V. Kermer, V. Baum,R.E. Kontermann, D. MullerAdministrative, technical, or material support: N. Hornig, V. KermerStudy supervision: D. Muller

Grant SupportThis work was supported by Deutsche Forschungsgemeinschaft (MU

2956/2-1).The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

Received January10, 2012; revisedFebruary 28, 2012; acceptedMarch 20,2012; published OnlineFirst April 6, 2012.

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2012;11:1279-1288. Published OnlineFirst April 6, 2012.Mol Cancer Ther Vanessa Kermer, Volker Baum, Nora Hornig, et al.

-Presentation at the Tumor SitetransIL-15 An Antibody Fusion Protein for Cancer Immunotherapy Mimicking

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