Combining Antibody-Directed Presentation of IL-15 and 4-1BBL in a Trifunctional Fusion Protein for Cancer Immunotherapy

Published OnlineFirst November 6, 2013; DOI: 10.1158/1535-7163.MCT-13-0282

Molecular Cancer Large Molecule Therapeutics Therapeutics

Vanessa Kermer, Nora Hornig, Markus Harder, Anastasiia Bondarieva, Roland E. Kontermann, and Dafne Muller€

Abstract Influencing the cytokine receptor network that modulates the immune response holds great potential for cancer immunotherapy. Although encouraging results have been obtained by focusing on individual members 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 proliferation 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 tumor mouse model in vivo. Hence, the targeted combination of IL-15 and 4-BBL in the form of a trifunctional antibody-fusion protein is a promising new approach for cancer immunotherapy. Mol Cancer Ther; 13(1); 1–10. 2013 AACR.

Introduction potential of IL-15 was shown by using IL-15 gene–mod- Immunomodulating cytokines have great potential in ified tumor cells (6), overexpression of IL-15 in transgenic cancer immunotherapy (1, 2). Interleukin (IL)-15 and 4- mice (7), or the administration of recombinant IL-15 in 1BB–directed agents have emerged here as promising several mouse models (8). In addition, the antitumor candidates (3). IL-15 belongs to the cytokines of the com- potential of recombinant IL-15 was shown to be further mon cytokine receptor g chain (gc) family (4). It is involved improved by complex formation with IL-15Ra-Fc (9) or in the generation, proliferation, and activation of natural the generation of a fusion protein with the fragment of the a killer (NK) cells, induces proliferation and differentiation IL-15R chain involved in ligand binding (extended sushi þ þ of CD8 T cells, and supports the survival of CD8 domain) (RD_IL-15; ref. 10). Also, a fusion protein com- memory T cells. Unlike IL-2, another clinical relevant posed of IL-15, IL-15Ra’s sushi domain, and apolipopro- member of this cytokine family, IL-15 rather inhibits tein A-I (Apo A-I) has been reported, inducing therapeutic activation-induced cell death (AICD) and seems not to effects in lung and liver metastases mouse models (11). influence regulatory T (Treg) cells (5). The antitumor Furthermore, tumor targeting by antibody fusion proteins with IL-15 (12) or RD_IL-15 (13, 14) resulted in enhanced antitumor effects in different mouse models. On the other hand, improved therapeutic effects could be achieved by Authors' Affiliation: Institut fur€ Zellbiologie und Immunologie, Universitat€ Stuttgart, Stuttgart, Germany the combination of IL-15 with other immunomodulatory approaches, for example, cytokines (IL-21, IL-7, IL-12; Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). refs. 6, 15, 16), agonistic anti-CD40 mAb (17), or inhibitory checkpoint blockers (anti-CTLA-4 mAb, anti-PD-L1 mAb; Corresponding Author: Dafne Muller,€ Institut fur€ Zellbiologie und Immu- nologie, Universitat€ Stuttgart, Allmandring 31, Stuttgart 70569, Germany. ref. 18). Thus, IL-15 holds great potential for combination Phone: 49-71168566999; Fax: 49-71168567484; E-mail: therapies. [email protected] 4-1BB is a costimulatory member of the TNF receptor doi: 10.1158/1535-7163.MCT-13-0282 superfamily (TNFR-SF) that is upregulated on activated T 2013 American Association for Cancer Research. cells and NK cells (19). Costimulation by 4-1BBL/4-1BB

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interaction is fundamentally involved in the proliferation, Materials and Methods þ differentiation, and survival of CD8 T cells, therefore Materials thought to play an important role in potentiating cytotoxic Antibodies and recombinant proteins were purchased T-cell immune responses (20). Antitumor effects were from Biolegends (mouse anti-human 4-1BBL-PE, rat anti- reported in several mouse models with agonistic 4- mouse 4-1BBL-PE, mouse anti-human CD3-PerCP), BD 1BB–specific antibodies (21, 22). Also, vaccination with Biosciences (mouse anti-human CD3-PE), Immunotools tumor cells transfected to express a 4-1BB–specific anti- (human recombinant IL-15), KPL [goat anti-mouse IgG body fragment (scFv) on the cell surface or admixture of (HþL)], Miltenyi Biotec (mouse anti-hexahistidyl-tag-PE), them to wild-type tumor cells induced strong antitumor R&D Systems (human 4-1BB-Fc, mouse 4-1BB-Fc, mouse responses (23, 24). Furthermore, antibody fusion proteins anti-human CD3e mAb), Santa Cruz (mouse anti-human with the extracellular domain of 4-1BBL showed target- CD107a-FITC), and Sigma (goat anti-human IgG(Fc)-PE). mediated costimulation in vitro (25, 26) and antitumor DuoSet ELISA kit for human IFN-g and CellTrace CFSE activity in vivo (27). Improved therapeutic effects were cell proliferation kit were obtained from R&D Systems achieved by the combination of 4-1BB costimulation with and Life Technologies, respectively. B16-FAP (transfec- diverse cytokines [e.g., IL-12, granulocyte macrophage tants with human FAP) and B16wt (K. Pfizenmaier, IZI) colony-stimulating factor (GM-CSF), IFN-a; refs. 28–30], were cultured in RPMI-1640, 5% FBS, supplemented with other costimulatory receptor activation (OX40, HVEM, 200 mg/mL zeocin in the case of B16-FAP. CTLL-2 cells (P. CD28; refs. 31–33), or inhibitory immune checkpoint Scheurich, IZI) were cultured in RPMI-1640, supplemen- blockade (anti-CTLA4 mAb; ref. 34). Thus, 4-1BB costi- ted with 20% FBS, 10 mmol/L HEPES, 0.05 mmol/L mulation seems particularly suitable for combination b-mercaptoethanol, 1 mmol/L natriumpyruvate, nones- therapies, too. sential amino acids, and 400 IU/mL rhIL-2. Cells were There is evidence that IL-15 and 4-1BBL act coopera- tested for mycoplasms and their morphologic appearance tively in the immune response. IL-15 was shown to induce monitored by microscopic means. Antigen (FAP) expres- þ 4-1BB expression on CD8 memory T cells in an antigen- sion on B16-FAP and cytokine growth dependence of independent manner, increasing their survival (35). Thus, CTLL-2 cells was verified by flow cytometry and prolif- both agents are linked in a model describing memory eration assays, respectively. Human peripheral blood þ CD8 T-cell homeostasis (36). On the other hand, success- mononuclear cells (PBMC) were isolated from buffy coat ful ex vivo expansion of human NK cells has been achieved of healthy donors [blood bank, Klinikum Stuttgart by stimulation with genetically modified K562 cells, (Katharinenhospital)] and cultivated in RPMI-1640, 10% expressing IL-15 and 4-1BBL. Hence, a potent and long- FBS. C57BL/6Jrj mice were purchased from Elevage Jan- lived NK cell population could be generated, capable to vier. Animal care and experiments carried out were in eradicate leukemia in a xenograft mouse model (37). accordance with federal guidelines and had been Therefore, immunotherapy involving the combination of approved by university and state authorities. IL-15 and 4-1BB costimulation should promote the induction of a strong antitumor response. Generation of recombinant antibody fusion proteins Here, we propose a targeted approach in form of a Generation of scFv_RD_IL-15 (13), scFv_4-1BBL (25), trifunctional antibody fusion protein to combine and and scDbFAPxCD3 (26) had been described previously. deliver IL-15 and 4-1BBL simultaneously to the tumor. RD_IL-15_scFv_4-1BBL was cloned starting from these The antibody is directed against the fibroblast activation bifunctional fusion proteins by introducing the human protein (FAP), a tumor stroma antigen expressed in RD_IL-15 N-terminally of the scFv_4-1BBL in the back- more than 90% of breast, colorectal, and lung carcino- bone vector pSecTagA (Life Technologies). RD_IL- mas (38). Targeting should hold advantages by modu- 15_scFv_m4-1BBL and scFv_m4-1BBL were obtained by lating the activity and accumulate the cytokines at the replacing the extracellular domain of human 4-1BBL (aa tumor site, thus reducing the effective dosage and the 71–254) by the corresponding mouse 4-1BBL (aa 104–309). risk of adverse events associated with a systemic admin- All recombinant proteins were produced in stably trans- istration. Moreover, application of the 2 cytokines in a fected HEK293 cells and were purified by immobilized single molecule format results in a spatiotemporal coor- metal ion affinity chromatography (IMAC) as described dinated activity, which is expected to focus the respec- elsewhere (13). In brief, producer cells were expanded and tive activity of the two cytokines on the same cell, grown to 90% confluence in RPMI 5% FBS before switch- ensuring cooperativity. Hence, an improvement of the ing to serum-free Opti-MEM I medium (Life Technolo- antitumor response could be expected. Therefore, we gies). Supernatants were collected and pooled. Proteins have generated a trifunctional fusion protein composed were concentrated by ammonium sulfate precipitation of a tumor-directed antibody in the scFv format, IL-15 (60% saturation), before loading onto a nickel nitrilotr- joint to an IL-15Ra chain fragment and the extracellular iacetic acid column (Qiagen) previously equilibrated domain of 4-1BBL. We investigated the immunostimu- with PBS. After a washing step with 50 mmol/L sodium latory capacity of this fusion protein format in vitro phosphate buffer, pH 7.5, 250 mmol/L NaCl, and 20 and evaluated its antitumor potential in a mouse model mmol/L imidazole, the recombinant fusion proteins were in vivo. eluted with 50 mmol/L sodium phosphate buffer, pH 7.5,

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250 mmol/L NaCl, and 250 mmol/L imidazole. Protein described for the PBMC proliferation assays was con- fractions were pooled and dialyzed against PBS. Integrity ducted. Here, supernatant was removed 5 days after and purity of the recombinant proteins were determined PBMC addition and concentration of IFN-g determined by SDS-PAGE and the identity was corroborated by by sandwich-ELISA following the instructions of the Western blotting. manufacturer’s protocol.

Binding analysis Cytotoxicity assay A total of 2 105 B16-FAP cells were incubated with The cytotoxic potential of T cells was assessed in terms each fusion protein for 2 hours at 4C. FAP-bound protein of degranulation and target cell killing. Fusion proteins at was detected by incubation for 1 hour at 4C with either different concentrations were incubated with 2 105 phycoerythrin (PE)-conjugated anti-hexahistidyl-tag anti- PBMCs per well for 5 days, either targeted to arrested body (scFv_RD_IL-15) or anti-4-1BBL as well as 4-1BB-Fc B16-FAP cells (see above) or without target cells. Then, followed by anti-huIgG(Fc) antibody, respectively PBMCs were transferred to a plate with freshly seeded (scFv_4-1BBL, RD_IL-15_scFv_4-1BBL). Washing and B16-FAP cells and T cells retargeted and triggered by incubation steps were carried out in PBS, 2% FBS, and incubation with 30 pmol/L bispecific antibody scDb 0.02% sodium azide. Cell analysis was conducted in an (FAPCD3) for 6 hours in the presence of 1.4 mL/well EPICS FC500 (Beckman Coulter), and data were analyzed GolgiStop (monensin, BD Biosciences). Subsequently, using FlowJo (Tree Star). PBMCs were harvested and T-cell degranulation measured by flow cytometry (CD107a-FITC/CD3-PerCP). In Proliferation assay with CTLL-2 parallel, T-cell cytotoxicity was determined, analyzing IL-15 activity was assessed on the cytokine growth- B16-FAP cell viability by MTT assay (13). dependent cell line CTLL-2. Therefore, 2 104 CTLL-2 cells per well were seeded and starved for 4 hours, before Animal experiment addition of the respective fusion proteins. After 3 days, Therapeutic efficacy of the recombinant proteins was cell proliferation was measured by MTT assay (13). assessed in a syngeneic B16-FAP lung metastasis mouse model. C57BL/6JRj mice (female, 4 months) were injected Proliferation assays with PBMCs i.v. with 8.5 105 B16-FAP cells per mouse on day 0. Human PBMCs were stained with carboxyfluorescein Treatment with (i) PBS, (ii) scFv_RD_IL-15, (iii) scFv_m4- diacetate succinimidyl ester (CFSE) at a concentration of 1BBL, and (iv) RD_IL-15_scFv_m4-1BBL (0.02 nmol 625 nmol/L/1 106 cells/mL, following the instructions fusion protein/animal) was administrated intraperitone- of the manufacturer. Subsequently, 2 105 PBMCs per ally (i.p.) on days 1, 2, and 10. Mice (6 mice/group) were well were applied in each assay and T-cell proliferation sacrificed on day 21. Lungs were removed, fixed in form- (anti-CD3-PE/CFSE) measured by flow cytometry. Pro- aldehyde, and metastases counted. liferation of PBMCs was assessed in response to the fusion proteins in targeted and nontargeted forms. In the former Statistic analysis case, 2 104 B16-FAP cells per well were seeded and For comparison of multiple groups, the one-way arrested the next day by incubating with mitomycin (10 ANOVA followed by the Tukey posttest was applied, mg/mL) for 2 hours at 37C. After washing, cells were using the GraphPad Prism software (GraphPad Soft- incubated for 1 hour at room temperature with the corre- ware). P < 0.05 was considered to be significant. sponding fusion protein at the indicated concentrations, followed by another washing step and the addition of Results PBMCs. Also, the effect of nontargeted fusion protein was Generation of the trifunctional fusion protein assessed. Therefore, B16wt cells were seeded and arrested Before the generation of the trifunctional fusion protein as indicated above. Then, fusion protein was added with human cytokines, the feasibility to improve immune together with PBMCs. In either case, blocking of the 4- cell response by combining IL-15 stimulation and 4-1BBL 1BBL–mediated effect of the trifunctional fusion protein costimulation had to be shown in vitro. Therefore, B16- (10 nmol/L) was achieved by addition of the recombinant FAP cells were cocultured with human PBMCs in the receptor 4-1BB-Fc (80 nmol/L). Furthermore, to analyze presence of cross-linked anti-human CD3 mAb, scFv_4- the effect on activated T cells, cross-linked anti-CD3 mAb 1BBL, and recombinant IL-15. Indeed, the combined (0.01 mg/mL) was added to the targeted and nontargeted application of human IL-15 and targeted human 4-1BBL setting. Cross-linking of the anti-CD3 mAb was achieved led to an enhancement in PBMC proliferation and IFN-g by previous incubation with goat anti-mouse IgG anti- release (Supplementary Fig. S1), providing a rational base bodies at a ratio of 1:3. to assess the immune-stimulating capacity of a corresponding trifunctional fusion protein. The trifunctional IFN-g release assays recombinant protein RD_IL-15_scFv_4-1BBL was gener- PBMC stimulation in response to fusion proteins was ated by the genetic fusion of human IL-15 (IL15) linked to also analyzed in terms of IFN-g release. Therefore, the an IL-15Ra (IL15RA) fragment (aa 31–107, containing the experimental setting with targeted fusion protein as IL-15 binding sushi domain), an antibody in the single

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ABFigure 1. A, schematic illustration RD_IL-15_scFv_4-1BBL of bi- and trifunctional fusion L20 L13 L14 L14 kDa proteins. scFv: single-chain Fv; RD IL-15 VH VL 4-1BBL (ECD) 170 VH,VL: variable region of heavy or 130 light antibody chain; RD: human 95 – 72 IL-15Ra (aa 31 107); IL-15: scFv_RD_IL-15 – 55 human IL-15 (aa 49 162); L14 L10 L20 4-1BBL(ECD): extracellular 43 VH VL RD IL-15 domain of human 4-1BBL 34 (aa 71–254) or mouse 4-1BBL (aa 104–309); L10:SG4SG4; scFv_4-1BBL 26 L13:G3SG3SSG3S; L14:G4 L14 L14 SG4SGGSA; L14 (with hexahistidyl-tag):

VH VL 4-1BBL (ECD) A3H6G4S; L20:G3 17 SG4SG3SG4SLQ; black box: hexahistidyl tag. B, characterization of the fusion proteins by 12% SDS-PAGE C under reducing conditions.

kDa Coomassie staining. C, high- performance liquid chromatography (HPLC) analysis of the fusion proteins on a TSK-GEL G3000SWXL column mAU mAU mAU (Tosoh Bioscience). Mobile phase 0.1 mol/L Na2SO4, 40 mmol/L Na2 HPO4, 60 mmol/L NaH2PO4,pH 6.7 at a ﬂow rate of 0.5 mL/min.

Time [min] Time [min] Time [min]

chain Fv (scFv) format directed against the FAP, a hex- (mouse/human) growth-dependent mouse cell line ahistidyl tag and the extracellular domain of human 4- CTLL-2, RD_IL-15_scFv_4-1BBL was observed to be 1BBL (TNFSF9; aa 71–254; Fig. 1A). As 4-1BBL is a member approximately 4-fold less active than scFv_RD_IL-15 (Fig. of the TNF superfamily, noncovalent homotrimer forma- 2C). As expected, no activity was measured for scFv_4- tion of the ligand and therefore the fusion protein was 1BBL (human 4-1BBL is not cross-reactive with mouse 4- expected, resulting in a molecule with 3 subunits of 1BB; Fig. 2C). Finally, ligand–receptor interaction of the RD_IL-15 and scFv, respectively. The bifunctional anti- human 4-1BBL compound with the corresponding human body fusion proteins used for reference were scFv_RD_IL- 4-1BB receptor was confirmed by flow cytometry (Fig. 15 and scFv_4-1BBL. They were also directed against FAP 2D). Therefore, binding of recombinant 4-1BB-Fc to target- (same antibody) and had been described previously bound RD_IL-15_scFv_4-1BBL and scFv_4-1BBL was (refs. 13, 25; Fig. 1A). Analysis by SDS-PAGE under detected via PE-conjugated anti-IgG(Fc) antibody. Thus, reducing conditions revealed a band of 76 kDa for all 3 components of the fusion protein RD_IL-15_scFv_4- RD_IL-15_scFv_4-1BBL (Fig. 1B). Considering predicted 1BBL proved to be functional. N-glycosylation for IL-15 at position 127, this correlates with the calculated molecular mass of 71 kDa of the In vitro activity of the trifunctional fusion protein monomer. Furthermore, size exclusion chromatography Human PBMC response was assessed in the presence of (SEC) of RD_IL-15_scFv_4-1BBL showed a main peak at the fusion protein in targeted and nontargeted forms on approximately 240 kDa, corroborating TNFSF ligand– B16-FAP cells and B16wt cells, respectively. PBMCs were mediated trimer formation (Fig. 1C). The latter was also applied either unstimulated or prestimulated with cross- shown for scFv_4-1BBL, whereas scFv_RD_IL-15 was linked anti-CD3 mAb. In the presence of CD3-stimulated present as a monomer and a small dimer fraction. PBMCs, proliferation could be efficiently enhanced by all 3 fusion proteins in targeted form (Fig. 3A). Here, Binding analysis and cytokine activity RD_IL-15_scFv_4-1BBL showed similar activity than Antibody-mediated binding of the fusion protein was scFv_RD_IL-15 and stronger activity than scFv_4-1BBL. analyzed by flow cytometry (Fig. 2A and B). Here, the In nontargeted form, the RD_IL-15_scFv_4-1BBL fusion trifunctional and the bifunctional fusion proteins showed protein was slightly less active than scFv_RD_IL-15 (Fig. similar binding capacity to FAP-expressing target cells 3B). As expected, nontargeted scFv_4-1BBL showed no (B16-FAP; Fig. 2B). No binding was detected on B16 wild- activity, consistent with previous reports (25). When type cells (Fig. 2A). In functional assays using the IL-15 experiments were carried out with unstimulated PBMCs,

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Figure 2. Functional properties of the antibody and cytokine components of the fusion proteins. A, antibody- binding specificity assessed on B16-FAP/B16-WT cells. B, comparing antibody-binding properties on B16-FAP cells. Bound fusion protein was detected via PE- conjugated monoclonal anti-4- 1BBL (RD_IL-15_scFv_4-1BBL, scFv_4-1BBL) or anti-His-tag antibody (scFv_RD_IL-15) by flow cytometry. C, IL-15 activity. Cytokine-dependent proliferation of CTLL-2 in the presence of the fusion proteins in solution was measured after 3 days by MTT assay. D, ligand–receptor interaction. B16- FAP cells were incubated with the fusion proteins (200 nmol/L), followed by incubation with the recombinant receptor 4-1BB-Fc (20 nmol/L). Ligand–receptor binding was detected via PE- conjugated anti-human Fc antibody by flow cytometry. Gray-filled, cells; black line, detection system; dotted line, antibody fusion protein. Graphics (B and C) show mean SD, n ¼ 3.

the IL-15 containing fusion proteins were able to initiate Fig. 4C), thus effectively promoting T-cell activation. The T-cell stimulation, whereas scFv_4-1BBL alone did not, participation of 4-1BBL in the signal enhancement was according to its costimulatory nature (i.e., requirement of shown by a blocking assay with recombinant 4-1BB-Fc ﬁrst signal; Fig. 4). In nontargeted form, RD_IL- receptor, reducing the proliferation induced by RD_IL- 15_scFv_4-1BBL and scFv_RD_IL-15 exerted at 10 15_scFv_4-1BBL to the level of that retrieved by nmol/L similar, but rather limited proliferation inducing scFv_RD_IL-15 only (Fig. 4D). Interestingly, the prolifer- activity (Fig. 4A). The situation changed completely, when ation induced by RD_IL-15_scFv_4-1BBL could not be the fusion proteins were presented in targeted form. Here, reached by the combination of the respective bifunctional RD_IL-15_scFv_4-1BBL was clearly more active than fusion proteins (scFv_RD_IL-15 and scFv_4-1BBL; Fig. scFv_RD_IL-15, inducing stronger T-cell proliferation 4D). In summary, on unstimulated PBMCs, the activity (up to 2.6-fold; Fig. 4B) and IFN-g release (up to 5.9-fold; of the trifunctional fusion protein in nontargeted form

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T cells were triggered and their cytotoxic potential determined by measuring T-cell degranulation and tumor cell killing. In targeted form, RD_IL-15_scFv_4-1BBL and scFv_RD_IL-15 showed comparable capacity in enhancing the cytotoxic potential of T cells (Fig. 5A), whereas in nontargeted form, at low concentrations, the effect of RD_IL-15_scFv_4-1BBL was slightly reduced in comparison to scFv_RD_IL-15 (Fig. 5B). In addition, the enhancement of the cytotoxic potential of the T-cell population showed to correlate with an increase in concomitant tumor cell killing, corroborating the antitumor potential of RD_IL-15_scFv_4-1BBL and scFv_RD-IL-15 in vitro (Fig. 5C,D).

In vivo activity of the trifunctional fusion protein To evaluate the antitumor potential of the RD_IL- 15_scFv_4-1BBL in a syngeneic tumor mouse model, the fusion protein had to be adjusted for mouse compatibility. Therefore, the extracellular domain of human 4-1BBL was replaced by the corresponding mouse domain (m4-1BBL). As RD_IL-15 is species cross-reactive in human and mice, no adjustment was necessary for this module of the fusion protein. The resulting trifunctional fusion protein RD_IL- 15_scFv_m4-1BBL retained binding and cytokine activity (Supplementary Fig. S2). The syngeneic B16 lung metastasis model has been used previously by us (13) and other groups (9, 10, 39) to evaluate immunotherapeutic treat- ments with IL-15–based reagents. B16-FAP cells were injected i.v. into C57BL/6 mice, and animals were treated with RD_IL-15_scFv_m4-1BBL, scFv_RD_IL-15, or scFv_m4-1BBL applied i.p. on days 1, 2, and 10. After 3 weeks, lungs were removed and tumor metastases counted. Significant antitumor effects were obtained with both, RD_IL-15_scFv_m4-1BBL and scFv_RD_IL-15 (Fig. 6). However, the best therapeutic result was obtained by treatment with the trifunctional molecule RD_IL- 15_scFv_m4-1BBL. Here, the number of metastases was reduced approximately to half the number of metastases Figure 3. Fusion protein–mediated effects on the proliferation of CD3- stimulated T cells. Fusion proteins were incubated on B16-FAP (A) or grown after treatment with scFv_RD_IL-15. Thus, super- B16wt (B) cells. After 1-hour incubation, cells were washed (A) or not (B) ior antitumor activity could be shown for the trifunctional before addition of cross-linked anti-CD3 mAb and CFSE-labeled PBMCs. fusion protein RD_IL-15_scFv_m4-1BBL in vivo. After 5 days, proliferation of T cells (CFSE/anti-CD3-PE) was analyzed by flow cytometry. Graphics show mean SD, n ¼ 23. Discussion We have reported here a trifunctional antibody fusion seems restricted to the IL-15 module, whereas 4-1BBL protein that combines a tumor-directed recombinant anti- remains inactive. The situation changes in the targeted body, RD_IL-15, and 4-1BBL into a single molecule. Each form, where 4-1BBL becomes active. Under these con- component retained its functionality, thus simultaneous ditions the 4-1BBL module enhances the IL-15 activity, antibody-mediated presentation of the IL and the costi- thus conferring higher immune stimulating activity to mulatory ligand of the TNF superfamily on target cells, the trifunctional fusion protein in comparison to the accompanied by their respective activity, was achieved. corresponding bifunctional IL-15 fusion protein. Previous studies with trifunctional antibody fusion pro- Furthermore, the impact of the fusion proteins on the teins had focused mainly on combinations involving the cytotoxic potential of T cells was analyzed (Fig. 5). There- cytokines IL-12, IL-2 and GM-CSF, using different tumor fore, unstimulated PBMCs were incubated for 5 days with targets (EpCAM, Her2/neu, CD30), antibody formats the fusion proteins either targeted to B16-FAP cells or (whole IgG, heterominibody, scFv-Fc), and cytokine com- without target cells. Subsequently, PBMCs were trans- binations (IL-2/IL-12, IL-12/GM-CSF, IL-2/GM-CSF; ferred to a fresh plate and T cells retargeted to B16-FAP refs. 40–44). Although activity of the respective antibody cells via a bispecific antibody (scDbFAPCD3). Thus, and cytokine components was shown in each case, the

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

Figure 4. Fusion protein–mediated effects on the proliferation and cytokine release of unstimulated T cells. Fusion proteins were incubated for 1 hour with B16-FAP (B, C, and D) or B16wt (A and D) cells. Then, cells were either washed (B16-FAP) or not (B16wt) and CFSE-labeled PBMCs added. After 5 days, proliferation of T cells (CFSE/anti-CD3-PE) was analyzed by ﬂow cytometry (A, B, and D) and IFN-g release was determined by sandwich ELISA (C). 4-1BBL– mediated contribution to the proliferation effect was determined by ligand-blocking assay (D). Therefore, the proliferation assay with targeted and untargeted fusion proteins (10 nmol/L) was conducted in the presence of 8-fold molar excess of the recombinant receptor 4-1BB-Fc. Graphics show mean SD, n ¼ 3. , P < 0.05; , P < 0.01; , P < 0.001.

antitumor effect of trifunctional and the respective bifunc- improved binding capacity of the antibody fusion protein tional antibody cytokine fusion proteins (alone or in in comparison to the scFv alone had been described pre- combination) was strongly dependent on the molecular viously (13). Higher avidity in terms of RD_IL-15 units design, cytokine combination, and tumor mouse model. seemed not to confer further advantages to the trifunctional Thus, not only the feasibility of the concept but also the molecule. Thus, in untargeted form, RD_IL-15_scFv_4- particular challenge of implementation became evident. 1BBL in comparison to scFv_RD_IL-15 showed similar or For members of the TNFSF, only a trifunctional antibody even lower activity on PBMC proliferation. This might be fusion protein with TNFa and IL-12 has been described so partially influenced by the position of the cytokine in the far (45). In this case, IL-12 was fused to the N-terminus and trifunctional molecule, as RD_IL-15_scFv in comparison to TNFa to the C-terminus of a scFv targeting the extrado- scFv_RD_IL-15 was recently shown to have slightly lower main B of fibronectin (ED-B). According to the structural activity on PBMC and CTLL-2 proliferation (Supplemen- properties of TNFa, assembling of a homotrimer was tary Fig. S3). Remarkably, in the trifunctional fusion pro- expected. Antibody binding and bioactivity of the fusion tein, 4-1BBL retained its characteristic bioactivity feature. protein in untargeted form was shown in vitro. Unfortu- Thus, in the nontargeted form of the fusion protein, the 4- nately, biodistribution studies with the fusion protein in 1BBL remained inactive and only in targeted form, that is, an immunocompetent 129SV mice bearing s.c. grafted F9 after scFv-mediated cell surface presentation, activity teratocarcinoma failed to show tumor accumulation and was observed in terms of an enhancement in IL-15–induced therefore further therapeutic studies were not attempted. T-cell proliferation and IFN-g release. This property has Thus, although an active fusion protein was engineered, been described for several other members of the TNFSF the functional requirements for the in vivo model were not (25, 46, 47) and was confirmed here for scFv_4-1BBL. fulfilled. Targeting-dependent acquisition of bioactivity might con- Here, we report for the first time a trifunctional antibody fer an advantage to the trifunctional fusion protein, con- fusion protein combining human 4-1BBL, another member sidering potential unwanted side effects of systemically of the TNFSF, and human IL-15, a member of the common active cytokines. gc receptor cytokine family, crucial for proliferation of Under physiologic conditions, 4-1BBL and IL-15 are effector T cells. Also in this case, TNFSF ligand–mediated presented on the cell surface, modulating the immune homotrimer formation was expected and confirmed. Anti- response upon cell–cell contact. Therefore, 4-1BBL is body-mediated targeting properties were not impaired by expressed as a transmembrane protein (48), whereas IL- the molecular design of this trifunctional fusion protein. 15 is presented in trans by the IL-15Ra chain on the cell Thus, the same functional affinity was shown for the surface (49). It was shown that the activity of IL-15 is trifunctional and the bifunctional, also homotrimeric strongly enhanced in the context of IL-15Ra chain pre- scFv_4-1BBL fusion protein, which was also similar to that sentation (50). Thus, IL-15IL15RaFc complexes or fusion of the mainly monomeric scFv_RD_IL-15. For the latter, proteins of IL-15 with the extended sushi domain of

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Figure 5. Fusion protein–mediated effects on T-cell cytotoxicity. Fusion proteins were incubated on B16-FAP cells, followed by washing and addition of PBMC (A and C). Alternatively, PBMCs were incubated with nontargeted fusion protein (B and D). After 5 days, PBMCs were transferred to a fresh plate with B16-FAP cells and T cells retargeted and triggered by the addition of 30 pmol/L scDbFAPCD3. After 6 hours, degranulating T cells were identiﬁed (anti-CD107a-FITC/ anti-CD3-PerCP) by ﬂow cytometry (A/B). In parallel, the cytotoxic effect on target cells 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.

IL-15Ra (RD) showed to be very effective in enhancing functional fusion protein of the present study. Thus, the NK cell and T-cell proliferation and increasing antitumor activity of RD_IL-15_scFv_4-1BBL and scFv_RD_IL-15 effects in diverse mouse models (9, 10, 39, 51). Further- were comparable in enhancing proliferation of activated more, tumor-directed antibody fusion proteins with T cells and promoting the expansion of cytotoxic T cells in RD_IL-15 showed improved antitumor effects in compar- vitro. The benefit of targeted 4-1BBL–mediated costimula- ison to untargeted RD_IL-15 in different mouse models tion became apparent under suboptimal activation con- (13, 14). The strong potential of RD_IL-15 was also ditions, that is, when the stimulatory activity of IL-15 was demonstrated and shown to be predominant in the tri- limited. Of note, simultaneous presentation of RD_IL-15 and 4-1BBL in one molecule was required, as the combination of equimolar amounts of the bifunctional antibody fusion proteins did not retrieve the cooperative effect observed for the targeted trifunctional antibody fusion protein. Thus, the targeted 2-in-1 cytokine fusion protein constellation apparently disposes of structural character- istics that favor cooperative activity. Encouraged by the antitumor effect observed in the animal experiment after treatment with the mouse compatible equivalent of the trifunctional fusion protein, following in vivo studies will have to focus now on the evaluation of the antitumor response of immune effector cell subpopulations as well as pharmacokinetic and pharmacodynamic properties of the fusion protein to determine the eligibility of such a trifunctional molecule for clinical translation. So far, clinical development focuses on recombinant IL-15 in phase I/II trials [acute myelogenous leukemia (AML), malig- Figure 6. Antitumor effect of fusion proteins analyzed in a lung metastasis nant melanoma, and solid tumors; ref. 2] and 2 agonistic mouse model. Mice were injected i.v. with B16-FAP cells on day 0. human 4-1BB–specific monoclonal antibodies (PF- Treatment of 0.02 nmol fusion protein/animal was applied i.p. on days 1, 05082566 and BMS-663513) in phase I (non–Hodgkin 2, and 10. After 21 days, lungs were removed and metastases counted. Number of metastases over 250 was considered uncountable and for lymphoma) and phase I/II (melanoma/solid tumors), graphic representation a fix value of 250 assigned. , P < 0.001. respectively (52).

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

Modulations of the immunologic conditions at the In summary, the feasibility and benefit of the trifunc- tumor site are gaining increasing interest, as it has been tional antibody fusion protein concept could be shown for recognized that solid tumors constitute an important the targeted combination of RD_IL-15 and 4-1BBL. These 2 place for the initiation and development of an antitumor cytokines display remarkably different structural and immune response. For example, it was shown in mouse functional properties that could be successfully bundled, þ models that na€ve CD8 T cells can infiltrate tumors and enhancing immune cell stimulation in vitro and antitumor become activated and differentiated into functional effec- response in vivo. tor cells in situ (53). Studies in mice provided evidence that fl localized activity of IL-15 and 4-1BB in the tumor micro- Disclosure of Potential Con icts of Interest R.E. Kontermann is a consultant/advisory board member of BioNTech. environment was relevant for the respective antitumor No potential conflicts of interest were disclosed by the other authors. response (54, 55). Thus, in a Rag1 / mouse model, IL-15– secreting tumors were grown in the presence of neutral- Authors' Contributions Conception and design: D. Muller izing IL-15 antibody and then after withdrawal of the Development of methodology: V. Kermer, D. Muller antibody, eradicated by an NK cell–mediated immune Acquisition of data (provided animals, acquired and managed patients, response. Co-expression of IL-15Ra by the tumor cells was provided facilities, etc.): V. Kermer, M. Harder, A. Bondarieva Analysis and interpretation of data (e.g., statistical analysis, biostatis- needed for the efficient induction of the densely granu- tics, computational analysis): V. Kermer, M. Harder, D. Muller lated NK effector cells in the tumor microenvironment. Writing, review, and/or revision of the manuscript: V. Kermer, N. Hornig, Moreover, the regression of the IL-15–secreting tumors R.E. Kontermann, D. Muller Administrative, technical, or material support (i.e., reporting or orga- did not stop the growth of contralateral non–IL-15-secret- nizing data, constructing databases): V. Kermer ing control tumors, suggesting that the effect of IL-15 was Study supervision: D. Muller largely restricted to the local microenvironment of the IL- Acknowledgments 15–secreting tumor (54). On the other hand, in another The authors thank Robert Lindner for technical support on the size mouse model, it was shown that the hypoxic conditions in exclusion chromatography analysis. the tumor induced the upregulation of 4-1BB on tumor- infiltrating lymphocytes (TIL), which could be selectively Grant Support activated by local, that is, intratumoral application of 4- This work was supported by a grant from the Deutsche Forschungsge- meinschaft (MU 2956/2-1; D. Muller).€ 1BB–specific monoclonal antibodies, promoting the The costs of publication of this article were defrayed in part by the development of an effective antitumor response without payment of page charges. This article must therefore be hereby marked causing liver side effects. (55). Thus, local copresentation advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. of IL-15 and 4-1BBL in the tumor microenvironment might be of especial value to support an antitumor Received April 15, 2013; revised September 27, 2013; accepted October immune response. 30, 2013; published OnlineFirst November 6, 2013.

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OF10 Mol Cancer Ther; 13(1) January 2014 Molecular Cancer Therapeutics

Combining Antibody-Directed Presentation of IL-15 and 4-1BBL in a Trifunctional Fusion Protein for Cancer Immunotherapy

Vanessa Kermer, Nora Hornig, Markus Harder, et al.

Mol Cancer Ther Published OnlineFirst November 6, 2013.

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