Combining Antibody-Directed Presentation of IL-15 and 4 ... · Generation of recombinant antibody fusion proteins Generation of scFv_RD_IL-15 (13), scFv_4-1BBL (25), and scDbFAPxCD3

Large Molecule Therapeutics

Combining Antibody-Directed Presentation of IL-15 and4-1BBL in a Trifunctional Fusion Protein for CancerImmunotherapy

Vanessa Kermer, Nora Hornig, Markus Harder, Anastasiia Bondarieva, Roland E. Kontermann, andDafne M€uller

AbstractInfluencing the cytokine receptor network that modulates the immune response holds great potential for

cancer immunotherapy.Although encouraging results have been obtained by focusing on individualmembers

of the common g-chain (gc) receptor family and TNF receptor superfamily so far, combination strategies might

be required to further improve the effectiveness of the antitumor response. Here, we propose the combination

of interleukin (IL)-15 and 4-1BBL in a single, tumor-directed molecule. Therefore, a trifunctional antibody

fusion protein was generated, composed of a tumor-specific recombinant antibody, IL-15 linked to a fragment

of the IL-15Ra chain (RD) and the extracellular domain of 4-1BBL. In soluble and targeted forms, the

trifunctional antibody fusion protein RD_IL-15_scFv_4-1BBL was shown to stimulate activated T-cell prolif-

eration and induce T-cell cytotoxicity to a similar degree as the bifunctional scFv_RD_IL-15 fusion protein. On

the other hand, in targeted form, the trifunctional fusion protein was much more effective in inducing T-cell

proliferation and IFN-g release of unstimulated peripheral blood mononuclear cells (PBMC). Here, the

additional signal enhancement could be attributed to the costimulatory activity of 4-1BBL, indicating a clear

benefit for the simultaneous presentation of IL-15 and 4-1BBL in one molecule. Furthermore, the trifunctional

antibody fusion protein was more effective than the corresponding bifunctional fusion proteins in reducing

metastases in a tumormousemodel in vivo. Hence, the targeted combination of IL-15 and 4-BBL in the formof a

trifunctional antibody-fusionprotein is apromisingnewapproach for cancer immunotherapy.MolCancerTher;

13(1); 112–21. �2013 AACR.

IntroductionImmunomodulating cytokines have great potential in

cancer immunotherapy (1, 2). Interleukin (IL)-15 and 4-1BB–directed agents have emerged here as promisingcandidates (3). IL-15 belongs to the cytokines of the com-mon cytokine receptor g chain (gc) family (4). It is involvedin the generation, proliferation, and activation of naturalkiller (NK) cells, induces proliferation and differentiationof CD8þ T cells, and supports the survival of CD8þ

memory T cells. Unlike IL-2, another clinical relevantmember of this cytokine family, IL-15 rather inhibitsactivation-induced cell death (AICD) and seems not toinfluence regulatory T (Treg) cells (5). The antitumor

potential of IL-15 was shown by using IL-15 gene–mod-ified tumor cells (6), overexpression of IL-15 in transgenicmice (7), or the administration of recombinant IL-15 inseveral mouse models (8). In addition, the antitumorpotential of recombinant IL-15 was shown to be furtherimproved by complex formation with IL-15Ra-Fc (9) orthe generation of a fusion proteinwith the fragment of theIL-15Ra chain involved in ligand binding (extended sushidomain) (RD_IL-15; ref. 10). Also, a fusion protein com-posed of IL-15, IL-15Ra’s sushi domain, and apolipopro-teinA-I (ApoA-I) has been reported, inducing therapeuticeffects in lung and liver metastases mouse models (11).Furthermore, tumor targeting by antibody fusionproteinswith IL-15 (12) or RD_IL-15 (13, 14) resulted in enhancedantitumor effects in different mousemodels. On the otherhand, improved therapeutic effects could be achieved bythe combination of IL-15 with other immunomodulatoryapproaches, for example, cytokines (IL-21, IL-7, IL-12;refs. 6, 15, 16), agonistic anti-CD40mAb (17), or inhibitorycheckpoint blockers (anti-CTLA-4mAb, anti-PD-L1mAb;ref. 18). Thus, IL-15 holds great potential for combinationtherapies.

4-1BB is a costimulatory member of the TNF receptorsuperfamily (TNFR-SF) that is upregulated on activated Tcells and NK cells (19). Costimulation by 4-1BBL/4-1BB

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

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

Corresponding Author: Dafne M€uller, Institut f€ur Zellbiologie und Immu-nologie, Universit€at Stuttgart, Allmandring 31, Stuttgart 70569, Germany.Phone: 49-71168566999; Fax: 49-71168567484; E-mail:[email protected]

doi: 10.1158/1535-7163.MCT-13-0282

�2013 American Association for Cancer Research.

MolecularCancer

Therapeutics

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on December 3, 2020. © 2014 American Association for Cancer Research. mct.aacrjournals.org Downloaded from

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interaction is fundamentally involved in the proliferation,differentiation, and survival of CD8þ T cells, thereforethought to play an important role in potentiating cytotoxicT-cell immune responses (20). Antitumor effects werereported in several mouse models with agonistic 4-1BB–specific antibodies (21, 22). Also, vaccination withtumor cells transfected to express a 4-1BB–specific anti-body fragment (scFv) on the cell surface or admixture ofthem to wild-type tumor cells induced strong antitumorresponses (23, 24). Furthermore, antibody fusion proteinswith the extracellular domain of 4-1BBL showed target-mediated costimulation in vitro (25, 26) and antitumoractivity in vivo (27). Improved therapeutic effects wereachieved by the combination of 4-1BB costimulation withdiverse cytokines [e.g., IL-12, granulocyte macrophagecolony-stimulating factor (GM-CSF), IFN-a; refs. 28–30],other costimulatory receptor activation (OX40, HVEM,CD28; refs. 31–33), or inhibitory immune checkpointblockade (anti-CTLA4 mAb; ref. 34). Thus, 4-1BB costi-mulation seems particularly suitable for combinationtherapies, too.There is evidence that IL-15 and 4-1BBL act coopera-

tively in the immune response. IL-15was shown to induce4-1BB expression on CD8þ memory T cells in an antigen-independentmanner, increasing their survival (35). Thus,both agents are linked in a model describing memoryCD8þ T-cell homeostasis (36). On the other hand, success-ful ex vivo expansion of humanNKcells has been achievedby stimulation with genetically modified K562 cells,expressing IL-15 and 4-1BBL. Hence, a potent and long-lived NK cell population could be generated, capable toeradicate leukemia in a xenograft mouse model (37).Therefore, immunotherapy involving the combination ofIL-15 and 4-1BB costimulation should promote the induc-tion of a strong antitumor response.Here, we propose a targeted approach in form of a

trifunctional antibody fusion protein to combine anddeliver IL-15 and 4-1BBL simultaneously to the tumor.The antibody is directed against the fibroblast activationprotein (FAP), a tumor stroma antigen expressed inmore than 90% of breast, colorectal, and lung carcino-mas (38). Targeting should hold advantages by modu-lating the activity and accumulate the cytokines at thetumor site, thus reducing the effective dosage and therisk of adverse events associated with a systemic admin-istration. Moreover, application of the 2 cytokines in asingle molecule format results in a spatiotemporal coor-dinated activity, which is expected to focus the respec-tive activity of the two cytokines on the same cell,ensuring cooperativity. Hence, an improvement of theantitumor response could be expected. Therefore, wehave generated a trifunctional fusion protein composedof a tumor-directed antibody in the scFv format, IL-15joint to an IL-15Ra chain fragment and the extracellulardomain of 4-1BBL. We investigated the immunostimu-latory capacity of this fusion protein format in vitroand evaluated its antitumor potential in a mouse modelin vivo.

Materials and MethodsMaterials

Antibodies and recombinant proteins were purchasedfrom Biolegends (mouse anti-human 4-1BBL-PE, rat anti-mouse 4-1BBL-PE, mouse anti-human CD3-PerCP), BDBiosciences (mouse anti-human CD3-PE), Immunotools(human recombinant IL-15), KPL [goat anti-mouse IgG(HþL)],MiltenyiBiotec (mouse anti-hexahistidyl-tag-PE),R&D Systems (human 4-1BB-Fc, mouse 4-1BB-Fc, mouseanti-human CD3e mAb), Santa Cruz (mouse anti-humanCD107a-FITC), and Sigma (goat anti-human IgG(Fc)-PE).DuoSet ELISA kit for human IFN-g and CellTrace CFSEcell proliferation kit were obtained from R&D Systemsand Life Technologies, respectively. B16-FAP (transfec-tants with human FAP) and B16wt (K. Pfizenmaier, IZI)were cultured in RPMI-1640, 5% FBS, supplemented with200 mg/mL zeocin in the case of B16-FAP. CTLL-2 cells (P.Scheurich, IZI) were cultured in RPMI-1640, supplemen-ted with 20% FBS, 10 mmol/L HEPES, 0.05 mmol/Lb-mercaptoethanol, 1 mmol/L natriumpyruvate, nones-sential amino acids, and 400 IU/mL rhIL-2. Cells weretested formycoplasms and their morphologic appearancemonitored by microscopic means. Antigen (FAP) expres-sion on B16-FAP and cytokine growth dependence ofCTLL-2 cells was verified by flow cytometry and prolif-eration assays, respectively. Human peripheral bloodmononuclear cells (PBMC) were isolated from buffy coatof healthy donors [blood bank, Klinikum Stuttgart(Katharinenhospital)] and cultivated in RPMI-1640, 10%FBS. C57BL/6Jrj mice were purchased from Elevage Jan-vier. Animal care and experiments carried out were inaccordance with federal guidelines and had beenapproved by university and state authorities.

Generation of recombinant antibody fusion proteinsGeneration of scFv_RD_IL-15 (13), scFv_4-1BBL (25),

and scDbFAPxCD3 (26) had been described previously.RD_IL-15_scFv_4-1BBL was cloned starting from thesebifunctional fusion proteins by introducing the humanRD_IL-15 N-terminally of the scFv_4-1BBL in the back-bone vector pSecTagA (Life Technologies). RD_IL-15_scFv_m4-1BBL and scFv_m4-1BBL were obtained byreplacing the extracellular domain of human 4-1BBL (aa71–254) by the correspondingmouse 4-1BBL (aa 104–309).All recombinant proteins were produced in stably trans-fected HEK293 cells and were purified by immobilizedmetal ion affinity chromatography (IMAC) as describedelsewhere (13). In brief, producer cellswere expandedandgrown to 90% confluence in RPMI 5% FBS before switch-ing to serum-free Opti-MEM I medium (Life Technolo-gies). Supernatants were collected and pooled. Proteinswere concentrated by ammonium sulfate precipitation(60% saturation), before loading onto a nickel nitrilotr-iacetic acid column (Qiagen) previously equilibratedwith PBS. After a washing step with 50 mmol/L sodiumphosphate buffer, pH 7.5, 250 mmol/L NaCl, and 20mmol/L imidazole, the recombinant fusion proteinswereelutedwith 50mmol/L sodium phosphate buffer, pH 7.5,

Combining IL-15 and 4-1BBL in an Antibody Fusion Protein

www.aacrjournals.org Mol Cancer Ther; 13(1) January 2014 113




250 mmol/L NaCl, and 250 mmol/L imidazole. Proteinfractions were pooled and dialyzed against PBS. Integrityand purity of the recombinant proteins were determinedby SDS-PAGE and the identity was corroborated byWestern blotting.

Binding analysisA total of 2 � 105 B16-FAP cells were incubated with

each fusion protein for 2 hours at 4�C. FAP-bound proteinwas detected by incubation for 1 hour at 4�C with eitherphycoerythrin (PE)-conjugated anti-hexahistidyl-tag anti-body (scFv_RD_IL-15) or anti-4-1BBL as well as 4-1BB-Fcfollowed by anti-huIgG(Fc) antibody, respectively(scFv_4-1BBL, RD_IL-15_scFv_4-1BBL). Washing andincubation steps were carried out in PBS, 2% FBS, and0.02% sodium azide. Cell analysis was conducted in anEPICS FC500 (Beckman Coulter), and data were analyzedusing FlowJo (Tree Star).

Proliferation assay with CTLL-2IL-15 activity was assessed on the cytokine growth-

dependent cell line CTLL-2. Therefore, 2 � 104 CTLL-2cells per well were seeded and starved for 4 hours, beforeaddition of the respective fusion proteins. After 3 days,cell proliferation was measured by MTT assay (13).

Proliferation assays with PBMCsHuman PBMCs were stained with carboxyfluorescein

diacetate succinimidyl ester (CFSE) at a concentration of625 nmol/L/1 � 106 cells/mL, following the instructionsof the manufacturer. Subsequently, 2 � 105 PBMCs perwell were applied in each assay and T-cell proliferation(anti-CD3-PE/CFSE) measured by flow cytometry. Pro-liferation of PBMCswas assessed in response to the fusionproteins in targeted and nontargeted forms. In the formercase, 2 � 104 B16-FAP cells per well were seeded andarrested the next day by incubating with mitomycin (10mg/mL) for 2 hours at 37�C. After washing, cells wereincubated for 1 hour at room temperature with the corre-sponding fusion protein at the indicated concentrations,followed by another washing step and the addition ofPBMCs. Also, the effect of nontargeted fusion proteinwasassessed. Therefore, B16wt cells were seeded and arrestedas indicated above. Then, fusion protein was addedtogether with PBMCs. In either case, blocking of the 4-1BBL–mediated effect of the trifunctional fusion protein(10 nmol/L) was achieved by addition of the recombinantreceptor 4-1BB-Fc (80 nmol/L). Furthermore, to analyzethe effect on activated T cells, cross-linked anti-CD3 mAb(0.01 mg/mL) was added to the targeted and nontargetedsetting. Cross-linking of the anti-CD3 mAb was achievedby previous incubation with goat anti-mouse IgG anti-bodies at a ratio of 1:3.

IFN-g release assaysPBMC stimulation in response to fusion proteins was

also analyzed in terms of IFN-g release. Therefore, theexperimental setting with targeted fusion protein as

described for the PBMC proliferation assays was con-ducted. Here, supernatant was removed 5 days afterPBMC addition and concentration of IFN-g determinedby sandwich-ELISA following the instructions of themanufacturer’s protocol.

Cytotoxicity assayThe cytotoxic potential of T cells was assessed in terms

of degranulation and target cell killing. Fusion proteins atdifferent concentrations were incubated with 2 � 105

PBMCs per well for 5 days, either targeted to arrestedB16-FAP cells (see above) or without target cells. Then,PBMCs were transferred to a plate with freshly seededB16-FAP cells and T cells retargeted and triggered byincubation with 30 pmol/L bispecific antibody scDb(FAP�CD3) for 6 hours in the presence of 1.4 mL/wellGolgiStop (monensin, BD Biosciences). Subsequently,PBMCs were harvested and T-cell degranulation mea-sured by flow cytometry (CD107a-FITC/CD3-PerCP). Inparallel, T-cell cytotoxicity was determined, analyzingB16-FAP cell viability by MTT assay (13).

Animal experimentTherapeutic efficacy of the recombinant proteins was

assessed in a syngeneic B16-FAP lung metastasis mousemodel. C57BL/6JRjmice (female, 4months) were injectedi.v. with 8.5 � 105 B16-FAP cells per mouse on day 0.Treatment with (i) PBS, (ii) scFv_RD_IL-15, (iii) scFv_m4-1BBL, and (iv) RD_IL-15_scFv_m4-1BBL (0.02 nmolfusion protein/animal) was administrated intraperitone-ally (i.p.) on days 1, 2, and 10. Mice (6 mice/group) weresacrificed on day 21. Lungs were removed, fixed in form-aldehyde, and metastases counted.

Statistic analysisFor comparison of multiple groups, the one-way

ANOVA followed by the Tukey posttest was applied,using the GraphPad Prism software (GraphPad Soft-ware). P < 0.05 was considered to be significant.

ResultsGeneration of the trifunctional fusion protein

Before the generation of the trifunctional fusion proteinwith human cytokines, the feasibility to improve immunecell response by combining IL-15 stimulation and 4-1BBLcostimulation had to be shown in vitro. Therefore, B16-FAP cells were cocultured with human PBMCs in thepresence of cross-linked anti-human CD3 mAb, scFv_4-1BBL, and recombinant IL-15. Indeed, the combinedapplication of human IL-15 and targeted human 4-1BBLled to an enhancement in PBMC proliferation and IFN-grelease (Supplementary Fig. S1), providing a rational baseto assess the immune-stimulating capacity of a corre-sponding trifunctional fusion protein. The trifunctionalrecombinant protein RD_IL-15_scFv_4-1BBL was gener-ated by the genetic fusion of human IL-15 (IL15) linked toan IL-15Ra (IL15RA) fragment (aa 31–107, containing theIL-15 binding sushi domain), an antibody in the single

Kermer et al.

Mol Cancer Ther; 13(1) January 2014 Molecular Cancer Therapeutics114




chain Fv (scFv) format directed against the FAP, a hex-ahistidyl tag and the extracellular domain of human 4-1BBL (TNFSF9; aa 71–254; Fig. 1A).As 4-1BBL is amemberof the TNF superfamily, noncovalent homotrimer forma-tion of the ligand and therefore the fusion protein wasexpected, resulting in a molecule with 3 subunits ofRD_IL-15 and scFv, respectively. The bifunctional anti-body fusionproteinsused for referencewere scFv_RD_IL-15 and scFv_4-1BBL. Theywere also directed against FAP(same antibody) and had been described previously(refs. 13, 25; Fig. 1A). Analysis by SDS-PAGE underreducing conditions revealed a band of 76 kDa forRD_IL-15_scFv_4-1BBL (Fig. 1B). Considering predictedN-glycosylation for IL-15 at position 127, this correlateswith the calculated molecular mass of 71 kDa of themonomer. Furthermore, size exclusion chromatography(SEC) of RD_IL-15_scFv_4-1BBL showed a main peak atapproximately 240 kDa, corroborating TNFSF ligand–mediated trimer formation (Fig. 1C). The latter was alsoshown for scFv_4-1BBL, whereas scFv_RD_IL-15 waspresent as a monomer and a small dimer fraction.

Binding analysis and cytokine activityAntibody-mediated binding of the fusion protein was

analyzed by flow cytometry (Fig. 2A and B). Here, thetrifunctional and the bifunctional fusion proteins showedsimilar binding capacity to FAP-expressing target cells(B16-FAP; Fig. 2B). No binding was detected on B16 wild-type cells (Fig. 2A). In functional assays using the IL-15

(mouse/human) growth-dependent mouse cell lineCTLL-2, RD_IL-15_scFv_4-1BBL was observed to beapproximately 4-fold less active than scFv_RD_IL-15 (Fig.2C). As expected, no activity was measured for scFv_4-1BBL (human 4-1BBL is not cross-reactive with mouse 4-1BB; Fig. 2C). Finally, ligand–receptor interaction of thehuman4-1BBL compoundwith the correspondinghuman4-1BB receptor was confirmed by flow cytometry (Fig.2D). Therefore, binding of recombinant 4-1BB-Fc to target-bound RD_IL-15_scFv_4-1BBL and scFv_4-1BBL wasdetected via PE-conjugated anti-IgG(Fc) antibody. Thus,all 3 components of the fusion protein RD_IL-15_scFv_4-1BBL proved to be functional.

In vitro activity of the trifunctional fusion proteinHumanPBMCresponsewas assessed in the presence of

the fusion protein in targeted and nontargeted forms onB16-FAP cells and B16wt cells, respectively. PBMCs wereapplied either unstimulated or prestimulated with cross-linked anti-CD3 mAb. In the presence of CD3-stimulatedPBMCs, proliferation could be efficiently enhanced byall 3 fusion proteins in targeted form (Fig. 3A). Here,RD_IL-15_scFv_4-1BBL showed similar activity thanscFv_RD_IL-15 and stronger activity than scFv_4-1BBL.In nontargeted form, the RD_IL-15_scFv_4-1BBL fusionprotein was slightly less active than scFv_RD_IL-15 (Fig.3B). As expected, nontargeted scFv_4-1BBL showed noactivity, consistent with previous reports (25). Whenexperiments were carried out with unstimulated PBMCs,

A B

C

RD_IL-15_scFv_4-1BBL

VH VLIL-15RD

L14 L14L20

4-1BBL (ECD)

L13

scFv_4-1BBL

Time [min]

mA

U

Time [min] Time [min]

VH VL

L14 L14

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VH VL IL-15RD

L10 L20L14

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55

43

34

26

17

95130170kDa

mA

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Figure 1. A, schematic illustrationof bi- and trifunctional fusionproteins. scFv: single-chain Fv;VH, VL: variable region of heavy orlight antibody chain; RD: humanIL-15Ra (aa 31–107); IL-15:human IL-15 (aa 49–162);4-1BBL(ECD): extracellulardomain of human 4-1BBL(aa 71–254) or mouse 4-1BBL(aa 104–309); L10: SG4SG4;L13: G3SG3SSG3S; L14: G4

SG4SGGSA; L14 (with hexahistidyl-tag):A3H6G4S; L20: G3

SG4SG3SG4SLQ; blackbox: hexahistidyl tag. B,characterization of the fusionproteins by 12% SDS-PAGEunder reducing conditions.Coomassie staining. C, high-performance liquidchromatography (HPLC) analysisof the fusion proteins on aTSK-GEL G3000SWXL column(Tosoh Bioscience). Mobile phase0.1 mol/L Na2SO4, 40 mmol/L Na2HPO4, 60 mmol/L NaH2PO4, pH6.7 at a flow rate of 0.5 mL/min.






the IL-15 containing fusion proteins were able to initiateT-cell stimulation, whereas scFv_4-1BBL alone did not,according to its costimulatory nature (i.e., requirement offirst signal; Fig. 4). In nontargeted form, RD_IL-15_scFv_4-1BBL and scFv_RD_IL-15 exerted at 10nmol/L similar, but rather limited proliferation inducingactivity (Fig. 4A). The situation changedcompletely,whenthe fusion proteinswere presented in targeted form.Here,RD_IL-15_scFv_4-1BBL was clearly more active thanscFv_RD_IL-15, inducing stronger T-cell proliferation(up to 2.6-fold; Fig. 4B) and IFN-g release (up to 5.9-fold;

Fig. 4C), thus effectively promoting T-cell activation. Theparticipation of 4-1BBL in the signal enhancement wasshown by a blocking assay with recombinant 4-1BB-Fcreceptor, reducing the proliferation induced by RD_IL-15_scFv_4-1BBL to the level of that retrieved byscFv_RD_IL-15 only (Fig. 4D). Interestingly, the prolifer-ation induced by RD_IL-15_scFv_4-1BBL could not bereached by the combination of the respective bifunctionalfusion proteins (scFv_RD_IL-15 and scFv_4-1BBL; Fig.4D). In summary, on unstimulated PBMCs, the activityof the trifunctional fusion protein in nontargeted form

Figure2. Functional properties of theantibody and cytokine componentsof the fusion proteins. A, antibody-binding specificity assessed onB16-FAP/B16-WT cells. B,comparing antibody-bindingproperties onB16-FAP cells. Boundfusion protein was detected via PE-conjugated monoclonal anti-4-1BBL (RD_IL-15_scFv_4-1BBL,scFv_4-1BBL) or anti-His-tagantibody (scFv_RD_IL-15) by flowcytometry. C, IL-15 activity.Cytokine-dependent proliferation ofCTLL-2 in the presence of the fusionproteins in solution was measuredafter 3 days by MTT assay. D,ligand–receptor interaction. B16-FAP cells were incubated with thefusion proteins (200 nmol/L),followed by incubation with therecombinant receptor 4-1BB-Fc(20 nmol/L). Ligand–receptorbinding was detected via PE-conjugated anti-human Fc antibodyby flow cytometry. Gray-filled, cells;black line, detection system; dottedline, antibody fusion protein.Graphics (B and C) show mean �SD, n ¼ 3.

Kermer et al.





seems restricted to the IL-15 module, whereas 4-1BBLremains inactive. The situation changes in the targetedform, where 4-1BBL becomes active. Under these con-ditions the 4-1BBL module enhances the IL-15 activity,thus conferring higher immune stimulating activity tothe trifunctional fusion protein in comparison to thecorresponding bifunctional IL-15 fusion protein.Furthermore, the impact of the fusion proteins on the

cytotoxic potential of T cells was analyzed (Fig. 5). There-fore, unstimulated PBMCswere incubated for 5 dayswiththe fusion proteins either targeted to B16-FAP cells orwithout target cells. Subsequently, PBMCs were trans-ferred to a fresh plate and T cells retargeted to B16-FAPcells via a bispecific antibody (scDbFAP�CD3). Thus,

T cells were triggered and their cytotoxic potential deter-mined by measuring T-cell degranulation and tumor cellkilling. In targeted form, RD_IL-15_scFv_4-1BBL andscFv_RD_IL-15 showed comparable capacity in enhanc-ing the cytotoxic potential of T cells (Fig. 5A), whereas innontargeted form, at low concentrations, the effect ofRD_IL-15_scFv_4-1BBL was slightly reduced in compar-ison to scFv_RD_IL-15 (Fig. 5B). In addition, the enhance-ment of the cytotoxic potential of the T-cell populationshowed to correlate with an increase in concomitanttumor cell killing, corroborating the antitumor potentialof RD_IL-15_scFv_4-1BBL and scFv_RD-IL-15 in vitro(Fig. 5C,D).

In vivo activity of the trifunctional fusion proteinTo evaluate the antitumor potential of the RD_IL-

15_scFv_4-1BBL in a syngeneic tumor mouse model, thefusion protein had to be adjusted formouse compatibility.Therefore, the extracellular domain of human 4-1BBLwasreplaced by the correspondingmouse domain (m4-1BBL).As RD_IL-15 is species cross-reactive in human andmice,no adjustmentwas necessary for thismodule of the fusionprotein. The resulting trifunctional fusion protein RD_IL-15_scFv_m4-1BBL retained binding and cytokine activity(Supplementary Fig. S2). The syngeneic B16 lung metas-tasis model has been used previously by us (13) and othergroups (9, 10, 39) to evaluate immunotherapeutic treat-ments with IL-15–based reagents. B16-FAP cells wereinjected i.v. into C57BL/6 mice, and animals were treatedwith RD_IL-15_scFv_m4-1BBL, scFv_RD_IL-15, orscFv_m4-1BBL applied i.p. on days 1, 2, and 10. After 3weeks, lungs were removed and tumor metastasescounted. Significant antitumor effects were obtained withboth, RD_IL-15_scFv_m4-1BBL and scFv_RD_IL-15 (Fig.6). However, the best therapeutic result was obtainedby treatment with the trifunctional molecule RD_IL-15_scFv_m4-1BBL. Here, the number of metastases wasreduced approximately to half the number of metastasesgrown after treatment with scFv_RD_IL-15. Thus, super-ior antitumor activity could be shown for the trifunctionalfusion protein RD_IL-15_scFv_m4-1BBL in vivo.

DiscussionWe have reported here a trifunctional antibody fusion

protein that combines a tumor-directed recombinant anti-body, RD_IL-15, and 4-1BBL into a single molecule. Eachcomponent retained its functionality, thus simultaneousantibody-mediated presentation of the IL and the costi-mulatory ligand of the TNF superfamily on target cells,accompanied by their respective activity, was achieved.Previous studies with trifunctional antibody fusion pro-teins had focused mainly on combinations involving thecytokines IL-12, IL-2 and GM-CSF, using different tumortargets (EpCAM, Her2/neu, CD30), antibody formats(whole IgG, heterominibody, scFv-Fc), and cytokine com-binations (IL-2/IL-12, IL-12/GM-CSF, IL-2/GM-CSF;refs. 40–44). Although activity of the respective antibodyand cytokine components was shown in each case, the

Figure 3. Fusion protein–mediated effects on the proliferation of CD3-stimulated T cells. Fusion proteins were incubated on B16-FAP (A) orB16wt (B) cells. After 1-hour incubation, cells were washed (A) or not (B)before addition of cross-linkedanti-CD3mAbandCFSE-labeledPBMCs.After 5 days, proliferation of T cells (CFSE/anti-CD3-PE) was analyzed byflow cytometry. Graphics show mean � SD, n ¼ 2�3.






antitumor effect of trifunctional and the respective bifunc-tional antibody cytokine fusion proteins (alone or incombination) was strongly dependent on the moleculardesign, cytokine combination, and tumor mouse model.Thus, not only the feasibility of the concept but also theparticular challenge of implementation became evident.For members of the TNFSF, only a trifunctional antibodyfusion proteinwith TNFa and IL-12 has been described sofar (45). In this case, IL-12was fused to theN-terminus andTNFa to the C-terminus of a scFv targeting the extrado-main B of fibronectin (ED-B). According to the structuralproperties of TNFa, assembling of a homotrimer wasexpected. Antibody binding and bioactivity of the fusionprotein in untargeted form was shown in vitro. Unfortu-nately, biodistribution studies with the fusion protein inan immunocompetent 129SV mice bearing s.c. grafted F9teratocarcinoma failed to show tumor accumulation andtherefore further therapeutic studies were not attempted.Thus, although an active fusion protein was engineered,the functional requirements for the in vivomodel were notfulfilled.

Here, we report for the first time a trifunctional antibodyfusion protein combining human 4-1BBL, anothermemberof the TNFSF, and human IL-15, a member of the commongc receptor cytokine family, crucial for proliferation ofeffector T cells. Also in this case, TNFSF ligand–mediatedhomotrimer formation was expected and confirmed. Anti-body-mediated targeting properties were not impaired bythe molecular design of this trifunctional fusion protein.Thus, the same functional affinity was shown for thetrifunctional and the bifunctional, also homotrimericscFv_4-1BBL fusion protein, which was also similar to thatof the mainly monomeric scFv_RD_IL-15. For the latter,

improved binding capacity of the antibody fusion proteinin comparison to the scFv alone had been described pre-viously (13). Higher avidity in terms of RD_IL-15 unitsseemednot to confer further advantages to the trifunctionalmolecule. Thus, in untargeted form, RD_IL-15_scFv_4-1BBL in comparison to scFv_RD_IL-15 showed similar oreven lower activity on PBMC proliferation. This might bepartially influenced by the position of the cytokine in thetrifunctionalmolecule, as RD_IL-15_scFv in comparison toscFv_RD_IL-15 was recently shown to have slightly loweractivity on PBMC and CTLL-2 proliferation (Supplemen-tary Fig. S3). Remarkably, in the trifunctional fusion pro-tein, 4-1BBL retained its characteristic bioactivity feature.Thus, in the nontargeted form of the fusion protein, the 4-1BBL remained inactive and only in targeted form, that is,after scFv-mediated cell surface presentation, activitywasobserved in termsofanenhancement in IL-15–inducedT-cell proliferation and IFN-g release. This property hasbeen described for several other members of the TNFSF(25, 46, 47) and was confirmed here for scFv_4-1BBL.Targeting-dependent acquisition of bioactivity might con-fer an advantage to the trifunctional fusion protein, con-sidering potential unwanted side effects of systemicallyactive cytokines.

Under physiologic conditions, 4-1BBL and IL-15 arepresented on the cell surface, modulating the immuneresponse upon cell–cell contact. Therefore, 4-1BBL isexpressed as a transmembrane protein (48), whereas IL-15 is presented in trans by the IL-15Ra chain on the cellsurface (49). It was shown that the activity of IL-15 isstrongly enhanced in the context of IL-15Ra chain pre-sentation (50). Thus, IL-15�IL15RaFc complexes or fusionproteins of IL-15 with the extended sushi domain of

Figure 4. Fusion protein–mediatedeffects on the proliferation andcytokine release of unstimulatedT cells. Fusion proteins wereincubated for 1 hour with B16-FAP(B, C, and D) or B16wt (A and D)cells. Then, cells were eitherwashed (B16-FAP) or not (B16wt)and CFSE-labeled PBMCs added.After 5 days, proliferation of T cells(CFSE/anti-CD3-PE) was analyzedby flow cytometry (A, B, and D) andIFN-g release was determinedby sandwich ELISA (C). 4-1BBL–mediated contribution to theproliferation effect was determinedby ligand-blocking assay (D).Therefore, the proliferation assaywith targeted and untargetedfusion proteins (10 nmol/L) wasconducted in the presence of8-fold molar excess of therecombinant receptor 4-1BB-Fc.Graphics show mean � SD,n ¼ 3. �, P < 0.05; ��, P < 0.01;��, P < 0.001.

Kermer et al.





IL-15Ra (RD) showed to be very effective in enhancingNK cell and T-cell proliferation and increasing antitumoreffects in diverse mouse models (9, 10, 39, 51). Further-more, tumor-directed antibody fusion proteins withRD_IL-15 showed improved antitumor effects in compar-ison to untargeted RD_IL-15 in different mouse models(13, 14). The strong potential of RD_IL-15 was alsodemonstrated and shown to be predominant in the tri-

functional fusion protein of the present study. Thus, theactivity of RD_IL-15_scFv_4-1BBL and scFv_RD_IL-15were comparable in enhancing proliferation of activatedT cells and promoting the expansion of cytotoxic T cells invitro. The benefit of targeted 4-1BBL–mediated costimula-tion became apparent under suboptimal activation con-ditions, that is, when the stimulatory activity of IL-15 waslimited. Of note, simultaneous presentation of RD_IL-15and 4-1BBL in one molecule was required, as the combi-nation of equimolar amounts of the bifunctional antibodyfusion proteins did not retrieve the cooperative effectobserved for the targeted trifunctional antibody fusionprotein. Thus, the targeted 2-in-1 cytokine fusion proteinconstellation apparently disposes of structural character-istics that favor cooperative activity. Encouraged by theantitumor effect observed in the animal experiment aftertreatment with the mouse compatible equivalent of thetrifunctional fusion protein, following in vivo studies willhave to focus now on the evaluation of the antitumorresponse of immune effector cell subpopulations as wellas pharmacokinetic and pharmacodynamic properties ofthe fusion protein to determine the eligibility of such atrifunctional molecule for clinical translation. So far, clin-ical development focuses on recombinant IL-15 in phaseI/II trials [acute myelogenous leukemia (AML), malig-nant melanoma, and solid tumors; ref. 2] and 2 agonistichuman 4-1BB–specific monoclonal antibodies (PF-05082566 and BMS-663513) in phase I (non–Hodgkinlymphoma) and phase I/II (melanoma/solid tumors),respectively (52).

Figure 5. Fusion protein–mediatedeffects on T-cell cytotoxicity.Fusion proteins were incubated onB16-FAP cells, followed bywashing and addition of PBMC(A and C). Alternatively, PBMCswere incubated with nontargetedfusion protein (B and D). After 5days, PBMCs were transferred to afresh plate with B16-FAP cells andT cells retargeted and triggered bythe addition of 30 pmol/LscDbFAP�CD3. After 6 hours,degranulating T cells wereidentified (anti-CD107a-FITC/anti-CD3-PerCP) by flowcytometry (A/B). In parallel,the cytotoxic effect on targetcells was shown by MTT assay(C and D). Graphics show mean �SD (A and B) and mean � SEM(C and D), n ¼ 3. �, P < 0.05;��, P < 0.01; ��, P < 0.001.

Figure 6. Antitumor effect of fusion proteins analyzed in a lungmetastasismouse model. Mice were injected i.v. with B16-FAP cells on day 0.Treatment of 0.02 nmol fusion protein/animal was applied i.p. on days 1,2, and 10. After 21 days, lungs were removed and metastases counted.Number of metastases over 250 was considered uncountable and forgraphic representation a fix value of 250 assigned. ��, P < 0.001.






Modulations of the immunologic conditions at thetumor site are gaining increasing interest, as it has beenrecognized that solid tumors constitute an importantplace for the initiation and development of an antitumorimmune response. For example, it was shown in mousemodels that na€�ve CD8þ T cells can infiltrate tumors andbecome activated and differentiated into functional effec-tor cells in situ (53). Studies inmice provided evidence thatlocalized activity of IL-15 and 4-1BB in the tumor micro-environment was relevant for the respective antitumorresponse (54, 55). Thus, in aRag1�/�mousemodel, IL-15–secreting tumors were grown in the presence of neutral-izing IL-15 antibody and then after withdrawal of theantibody, eradicated by an NK cell–mediated immuneresponse.Co-expressionof IL-15Raby the tumor cellswasneeded for the efficient induction of the densely granu-lated NK effector cells in the tumor microenvironment.Moreover, the regression of the IL-15–secreting tumorsdid not stop the growth of contralateral non–IL-15-secret-ing control tumors, suggesting that the effect of IL-15 waslargely restricted to the local microenvironment of the IL-15–secreting tumor (54). On the other hand, in anothermousemodel, it was shown that the hypoxic conditions inthe tumor induced the upregulation of 4-1BB on tumor-infiltrating lymphocytes (TIL), which could be selectivelyactivated by local, that is, intratumoral application of 4-1BB–specific monoclonal antibodies, promoting thedevelopment of an effective antitumor response withoutcausing liver side effects. (55). Thus, local copresentationof IL-15 and 4-1BBL in the tumor microenvironmentmight be of especial value to support an antitumorimmune response.

In summary, the feasibility and benefit of the trifunc-tional antibody fusion protein concept could be shown forthe targeted combination ofRD_IL-15 and4-1BBL. These 2cytokines display remarkably different structural andfunctional properties that could be successfully bundled,enhancing immune cell stimulation in vitro and antitumorresponse in vivo.

Disclosure of Potential Conflicts of InterestR.E. Kontermann is a consultant/advisory boardmember of BioNTech.

No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: D. MullerDevelopment of methodology: V. Kermer, D. MullerAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): V. Kermer, M. Harder, A. BondarievaAnalysis and interpretation of data (e.g., statistical analysis, biostatis-tics, computational analysis): V. Kermer, M. Harder, D. MullerWriting, review, and/or revisionof themanuscript:V.Kermer,N.Hornig,R.E. Kontermann, D. MullerAdministrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): V. KermerStudy supervision: D. Muller

AcknowledgmentsThe authors thank Robert Lindner for technical support on the size

exclusion chromatography analysis.

Grant SupportThis work was supported by a grant from the Deutsche Forschungsge-

meinschaft (MU 2956/2-1; D. M€uller).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 April 15, 2013; revised September 27, 2013; accepted October30, 2013; published OnlineFirst November 6, 2013.

References1. PardollD,DrakeC. Immunotherapy earns its spot in the ranksof cancer

therapy. J Exp Med 2012;209:201–9.2. Kontermann RE. Antibody-cytokine fusion proteins. Arch Biochem

Biophys 2012;526:194–205.3. Cheever MA. Twelve immunotherapy drugs that could cure cancers.

Immunol Rev 2008;222:357–68.4. Rochman Y, Spolski R, Leonard WJ. New insights into the regulation

of T cells by gamma(c) family cytokines. Nat Rev Immunol 2009;9:480–90.

5. Steel JC, Waldmann TA, Morris JC. Interleukin-15 biology and itstherapeutic implications in cancer. Trends Pharmacol Sci 2012;33:35–41.

6. Kimura K, Nishimura H, Matsuzaki T, Yokokura T, Nimura Y, YoshikaiY. Synergistic effect of interleukin-15 and interleukin-12 on antitumoractivity in amurinemalignant pleurisymodel. Cancer Immunol Immun-other 2000;49:71–7.

7. YajimaT,NishimuraH,WajjwalkuW,HaradaM,KuwanoH,Yoshikai Y.Overexpression of interleukin-15 in vivo enhances antitumor activityagainst MHC class I-negative and -positive malignant melanomathrough augmented NK activity and cytotoxic T-cell response. Int JCancer 2002;99:573–8.

8. Tang F, Zhao LT, Jiang Y, Ba de N, Cui LX, He W. Activity ofrecombinant human interleukin-15 against tumor recurrence andmetastasis in mice. Cell Mol Immunol 2008;5:189–96.

9. Dubois S, Patel HJ, ZhangM,WaldmannTA,M€uller JR. Preassociationof IL-15 with IL-15R alpha-IgG1-Fc enhances its activity on prolifer-ation of NK and CD8þ/CD44high T cells and its antitumor action.J Immunol 2008;180:2099–106.

10. Bessard A, Sol�e V, Bouchaud G, Qu�em�ener A, Jacques Y. Highantitumor activity of RLI, an interleukin-15 (IL-15)-IL-15 receptor alphafusion protein, in metastatic melanoma and colorectal cancer. MolCancer Ther 2009;8:2736–45.

11. Ochoa MC, Fioravanti J, Rodriguez I, Hervas-Stubbs S, Azpilikueta A,Mazzolini G, et al. Antitumor immunotherapeutic and toxic propertiesof an HDL-conjugated chimeric IL-15 fusion protein. Cancer Res2013;73:139–49.

12. Kaspar M, Trachsel E, Neri D. The antibody-mediated targeteddelivery of interleukin-15 and GM-CSF to the tumor neovasculatureinhibits tumor growth and metastasis. Cancer Res 2007;67:4940–8.

13. Kermer V, Baum V, Hornig N, Kontermann RE, M€uller D. An antibodyfusion protein for cancer immunotherapy mimicking IL-15 trans-pre-sentation at the tumor site. Mol Cancer Ther 2012;11:1279–88.

14. VincentM,BessardA,CochonneauD, TeppazG,Sol�eV,MaillassonM,et al. Tumor targeting of the IL-15 superagonist RLI by an anti-GD2antibody strongly enhances its antitumor potency. Int J Cancer2013;133:757–65.

15. Kishida T, Asada H, Itokawa Y, Cui FD, Shin-Ya M, Gojo S, et al.Interleukin (IL)-21 and IL-15 genetic transfer synergistically augmentstherapeutic antitumor immunity andpromotes regressionofmetastaticlymphoma. Mol Ther 2003;8:552–8.

16. Habibi M, Kmieciak M, Graham L, Morales JK, Bear HD, Manjili MH.Radiofrequency thermal ablation of breast tumors combined withintralesional administration of IL-7 and IL-15 augments anti-tumorimmune responses and inhibits tumor development and metastasis.Breast Cancer Res Treat 2009;114:423–31.

Kermer et al.





17. ZhangM, Yao Z, Dubois S, JuW,M€uller JR,Waldmann TA. Interleukin-15 combined with an anti-CD40 antibody provides enhanced thera-peutic efficacy formurinemodels of colon cancer. ProcNatl AcadSciUS A 2009;106:7513–8.

18. Yu P, Steel JC, Zhang M, Morris JC, Waldmann TA. Simultaneousblockade of multiple immune system inhibitory checkpointsenhances antitumor activity mediated by interleukin-15 in a murinemetastatic colon carcinoma model. Clin Cancer Res 2010;16:6019–28.

19. Cheuk AT, Mufti GJ, Guinn BA. Role of 4-1BB:4-1BB ligand in cancerimmunotherapy. Cancer Gene Ther 2004;11:215–26.

20. Wang C, Lin GH, McPherson AJ, Watts TH. Immune regulation by 4-1BB and 4-1BBL: complexities and challenges. Immunol Rev2009;229:192–215.

21. Melero I, Shuford WW, Newby SA, Aruffo A, Ledbetter JA,Hellstr€om KE, et al. Monoclonal antibodies against the 4-1BBT-cell activation molecule eradicate established tumors. Nat Med1997;3:682–5.

22. Fisher TS, Kamperschroer C, Oliphant T, Love VA, Lira PD, DoyonnasR, et al. Targeting of 4-1BB by monoclonal antibody PF-05082566enhances T-cell function and promotes anti-tumor activity. CancerImmunol Immunother 2012;61:1721–33.

23. Ye Z, Hellstr€om I, Hayden-Ledbetter M, Dahlin A, Ledbetter JA,Hellstr€omKE.Gene therapy for cancer using single-chain Fv fragmentsspecific for 4-1BB. Nat Med 2002;8:343–8.

24. Yang Y, Yang S, Ye Z, Jaffar J, Zhou Y, Cutter E, et al. Tumor cellsexpressing anti-CD137 scFv induce a tumor-destructive environment.Cancer Res 2007;67:2339–44.

25. M€uller D, Frey K, Kontermann RE. A novel antibody-4-1BBL fusionprotein for targeted costimulation in cancer immunotherapy. J Immun-other 2008;31:714–22.

26. Hornig N, Kermer V, Frey K, Diebolder P, Kontermann RE, M€uller D.Combination of a bispecific antibody and costimulatory antibody-ligand fusion proteins for targeted cancer immunotherapy. J Immun-other 2012;35:418–29.

27. Zhang N, Sadun RE, Arias RS, Flanagan ML, Sachsman SM, Nien YC,et al. Targeted and untargeted CD137L fusion proteins for the immu-notherapy of experimental solid tumors. Clin Cancer Res 2007;13:2758–67.

28. Li B, Lin J, Vanroey M, Jure-Kunkel M, Jooss K. Established B16tumors are rejected following treatmentwith GM-CSF-secreting tumorcell immunotherapy in combination with anti-4-1BB mAb. Clin Immu-nol 2007;125:76–87.

29. Dubrot J, Palaz�on A, Alfaro C, Azpilikueta A, Ochoa MC, Rouzaut A,et al. Intratumoral injection of interferon-a and systemic delivery ofagonist anti-CD137monoclonal antibodies synergize for immunother-apy. Int J Cancer 2011;128:105–18.

30. Martinet O, Divino CM, Zang Y, Gan Y, Mandeli J, Thung S, et al. T cellactivation with systemic agonistic antibody versus local 4-1BB ligandgene delivery combined with interleukin-12 eradicate liver metastasesof breast cancer. Gene Ther 2002;9:786–92.

31. Gray JC, French RR, James S, Al-Shamkhani A, Johnson PW,Glennie MJ. Optimising anti-tumour CD8 T-cell responses usingcombinations of immunomodulatory antibodies. Eur J Immunol2008;38:2499–511.

32. Park JJ, Anand S, Zhao Y, Matsumura Y, Sakoda Y, Kuramasu A, et al.Expression of anti-HVEMsingle-chain antibody on tumor cells inducestumor-specific immunity with long-term memory. Cancer ImmunolImmunother 2012;61:203–14.

33. Li G, Wu X, Zhang F, Li X, Sun B, Yu Y, et al. Triple expression of B7-1,B7-2 and 4-1BBL enhanced antitumor immune response againstmouse H22 hepatocellular carcinoma. J Cancer Res Clin Oncol2011;137:695–703.

34. Kocak E, Lute K, Chang X, May KF Jr, Exten KR, Zhang H, et al.Combination therapy with anti-CTL antigen-4 and anti-4-1BB anti-bodies enhances cancer immunity and reduces autoimmunity. CancerRes 2006;66:7276–84.

35. Pulle G, Vidric M, Watts TH. IL-15-dependent induction of 4-1BBpromotes antigen-independentCD8memory T cell survival. J Immunol2006;176:2739–48.

36. Sabbagh L, Snell LM, Watts TH. TNF family ligands define niches for Tcell memory. Trends Immunol 2007;28:333–9.

37. Fujisaki H, Kakuda H, Shimasaki N, Imai C, Ma J, Lockey T , et al.Expansion of highly cytotoxic human natural killer cells for cancer celltherapy. Cancer Res 2009;69:4010–7.

38. Garin-Chesa P, Old LJ, RettigWJ. Cell surface glycoprotein of reactivestromal fibroblasts as a potential antibody target in human epithelialcancers. Proc Natl Acad Sci U S A 1990;87:7235–9.

39. Stoklasek TA, Schluns KS, Lefrancois L. Combined IL-15/IL-15Ralphaimmunotherapy maximizes IL-15 activity in vivo. J Immunol 2006;177:6072–80.

40. Gillies SD, Lan Y, Brunkhorst B, Wong WK, Li Y, Lo KM. Bifunctionalcytokine fusion proteins for gene therapy and antibody-targeted treat-ment of cancer. Cancer Immunol Immunother 2002;51:449–60.

41. HelgueraG,Rodríguez JA, PenichetML.Cytokines fused to antibodiesand their combinations as therapeutic agents against different peri-toneal HER2/neu expressing tumors. Mol Cancer Ther 2006;5:1029–40.

42. Schanzer JM,Baeuerle PA,Dreier T, Kufer P. A humancytokine/single-chain antibody fusion protein for simultaneous delivery of GM-CSFand IL-2 to Ep-CAM overexpressing tumor cells. Cancer Immun2006;6:4.

43. Schanzer JM, Fichtner I, Baeuerle PA, Kufer P. Antitumor activity of adual cytokine/single-chain antibody fusion protein for simultaneousdelivery of GM-CSF and IL-2 to Ep-CAM expressing tumor cells.J Immunother 2006;29:477–88.

44. Jahn T, Zuther M, Friedrichs B, Heuser C, Guhlke S, Abken H, et al. AnIL12-IL2-antibody fusion protein targeting Hodgkin's lymphoma cellspotentiates activation of NK and T cells for an anti-tumor attack. PLoSOne 2012;7:e44482.

45. Halin C, Gafner V, Villani ME, Borsi L, Berndt A, Kosmehl H, et al.Synergistic therapeutic effects of a tumor targeting antibody fragment,fused to interleukin 12 and to tumor necrosis factor alpha. Cancer Res2003;63:3202–10.

46. Wajant H, Gerspach J, Pfizenmaier K. Tumor therapeutics by design:targeting and activation of death receptors. Cytokine Growth FactorRev 2005;16:55–76.

47. M€uller N, Wyzgol A, M€unkel S, Pfizenmaier K, Wajant H. Activity ofsoluble OX40 ligand is enhanced by oligomerization and cell surfaceimmobilization. FEBS J 2008;275:2296–304.

48. Bodmer JL, Schneider P, Tschopp J. Themolecular architecture of theTNF superfamily. Trends Biochem Sci 2002;27:19–26.

49. Dubois S, Mariner J, Waldmann TA, Tagaya Y. IL-15Ralpha recyclesand presents IL-15 In trans to neighboring cells. Immunity 2002;17:537–47.

50. RubinsteinMP, KovarM, Purton JF,Cho JH,BoymanO,SurhCD, et al.Converting IL-15 to a superagonist by binding to soluble IL-15R{alpha}. Proc Natl Acad Sci U S A 2006;103:9166–71.

51. Huntington ND, Alves NL, Legrand N, Lim A, Strick-Marchand H,Mention JJ, et al. IL-15 transpresentation promotes both human T-cell reconstitution and T-cell-dependent antibody responses in vivo.Proc Natl Acad Sci U S A 2011;108:6217–22.

52. CroftM, Benedict CA,WareCF. Clinical targeting of the TNF and TNFRsuperfamilies. Nat Rev Drug Discov 2013;12:147–68.

53. Thompson ED, Enriquez HL, Fu YX, Engelhard VH. Tumor massessupport naive T cell infiltration, activation, and differentiation intoeffectors. J Exp Med 2010;207:1791–804.

54. Liu RB, Engels B, Arina A, Schreiber K, Hyjek E, Schietinger A, et al.Densely granulated murine NK cells eradicate large solid tumors.Cancer Res 2012;72:1964–74.

55. Palaz�on A, Martínez-Forero I, Teijeira A, Morales-Kastresana A, AlfaroC, Sanmamed MF, et al. The HIF-1a hypoxia response in tumor-infiltrating T lymphocytes induces functional CD137 (4-1BB) for immu-notherapy. Cancer Discov 2012;2:608–23.






2014;13:112-121. Published OnlineFirst November 6, 2013.Mol Cancer Ther Vanessa Kermer, Nora Hornig, Markus Harder, et al. Trifunctional Fusion Protein for Cancer ImmunotherapyCombining Antibody-Directed Presentation of IL-15 and 4-1BBL in a

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