ORIGINAL RESEARCH OncoImmunology 4:9, e1038011; September 2015; © 2015 Taylor & Francis Group, LLC Induction of potent NK cell-dependent anti-myeloma cytotoxic T cells in response to combined mapatumumab and

Paul J Neeson1,2,3,*,y, Andy K Hsu1,2,y, Yin R Chen1,2, Heloise M Halse1,2, Joanna Loh1,2, Reece Cordy1,2, Kate Fielding1,2, Joanne Davis1,2,3,4, Josh Noske1,2, Alex J Davenport1,2,4, Camilla A Lindqvist-Gigg1,2,4, Robin Humphreys5, Tsin Tai1,2, H Miles Prince2,6, Joseph A Trapani1,2,3, Mark J Smyth2,7,8, and David S Ritchie1,2,3,4,9

1Cancer Immunology Research; Peter MacCallum Cancer Center; East Melbourne, VIC Australia; 2Sir Peter MacCallum Department of Oncology; University of Melbourne; Parkville, VIC Australia; 3The University of Melbourne; Parkville, VIC Australia; 4The ACRF Translational Research Laboratory; Royal Melbourne Hospital; Parkville, VIC Australia; 5Human Genome Sciences Inc.; Rockville, MD USA; 6Department of Cancer Medicine; Peter MacCallum Cancer Center; East Melbourne, VIC Australia; 7Immunology in Cancer and Infection Laboratory; Queensland Institute of Medical Research; Herston, QLD Australia; 8School of Medicine; University of Queensland; Herston, Australia; 9Department of Clinical Hematology and Bone Marrow Transplantation; Royal Melbourne Hospital; Parkville, VIC Australia

yIndicates equal first authorship.

Keywords: dendritic cell, , natural killer cell, proteasome inhibitor, T cell, apoptosis inducing ligand and receptor Abbreviations: anti-myeloma cytotoxic lymphocyte, AMCL; bortezomib, btz; chymotrypsin, Ch; cytotoxic lymphocyte, CTL; dendritic cell, DC; human myeloma cell line, HMCL; low dose bortezomib, LDB; low dose bortezomib and mapatumumab, LDBCMapa; mapatumumab, Mapa; monocyte-derived dendritic cell, MoDC; Multiple myeloma, MM; TRAIL receptor 1, TRAIL-R1; tumor necrosis factor apoptosis inducing ligand, TRAIL

There is increasing evidence that some cancer therapies can promote tumor immunogenicity to boost the endogenous antitumor immune response. In this study, we used the novel combination of agonistic anti-TRAIL-R1 antibody (mapatumumab, Mapa) with low dose bortezomib (LDB) for this purpose. The combination induced profound myeloma cell apoptosis, greatly enhanced the uptake of myeloma cell apoptotic bodies by dendritic cell (DC) and induced anti-myeloma cytotoxicity by both CD8C T cells and NK cells. Cytotoxic lymphocyte expansion was detected within 24 h of commencing therapy and was maximized when myeloma-pulsed DC were co-treated with low dose bortezomib and mapatumumab (LDBCMapa) in the presence of NK cells. This study shows that Mapa has two distinct but connected modes of action against multiple myeloma (MM). First, when combined with LDB, Mapa produced powerful myeloma cell apoptosis; secondly, it promoted DC priming and an NK cell-mediated expansion of anti- myeloma cytotoxic lymphocyte (CTL). Overall, this study indicates that Mapa can be used to drive potent anti-MM immune responses.

Introduction trials17-20 suggest that antibodies directed against plasma cells including those against SLAM-F7 (, Bristol-Myers MM is incurable by either conventional chemotherapy or Squibb) or CD38 (, Janssen Biotech) have the recently developed novel therapies including immunomodulatory potential to significantly improve immunotherapy against MM. drugs and proteasome inhibitors.1,2,3 This underscores the need The effect of these antibodies at promoting secondary cytotoxic for the further development of therapies that target all aspects of cellular immune responses has not yet been full explored. Mono- myeloma control including cell survival, proliferation4 and anti- clonal antibodies directed against the tumor necrosis factor- myeloma immunity.5 MM is associated with defects in both cel- related apoptosis-inducing ligand receptor-1 (TRAIL-R1) may lular and humoral immunity6-9 that must be overcome to opti- also offer an opportunity to induce myeloma cell apoptosis, pro- mize immunotherapeutic approaches.10 Ideal anti-MM therapy mote immunogenic cell death and generate cytotoxic cellular would be that which delivers maximal myeloma cell apoptosis immune responses. whilst simultaneously promoting anti-myeloma immune Tumor necrosis factor-related apoptosis-inducing ligand responses by promoting immunogenic cell death.11 Recent stud- (TRAIL or Apo2L) induces apoptosis by activation of either ies in pre-clinical models,12-16 and promising results in clinical TRAIL-R1 (DR4) or TRAIL-R2 (DR5).21-24 Soluble TRAIL

*Correspondence to: Paul J Neeson; Email: [email protected] Submitted: 07/18/2014; Revised: 03/30/2015; Accepted: 04/01/2015 http://dx.doi.org/10.1080/2162402X.2015.1038011 www.tandfonline.com OncoImmunology e1038011-1 induces apoptotic cell death in a wide range of tumors, including (Fig. S2D), consistent with that observed previously by Menoret MM, but not in normal cells, however its therapeutic utility is et al. 25 Thus, whilst OPM-2 was sensitive to Mapa therapy, limited by its relatively short half-life. The agonistic humanized OPM-2 constitutively expressed relatively lower levels of TRAIL- IgG1 antibody to TRAIL-R1, Mapa, has been previously shown R1 than LP-1, a low responder to Mapa-induced apoptosis. to induce apoptosis in MM cells;25 however, the immune poten- Subsequently, all six MM cell lines were treated with LDB tiating effect of this antibody has not been previously explored. (established above for each cell line) and titrated doses of Mapa The combination of TRAIL ligand and bortezomib (btz) has to establish whether combination therapy was more effective been shown to induce apoptosis in MM cell lines and primary than Mapa alone. RPMI8226, OPM-2 and U266 were all signifi- human samples.26 Furthermore, Mapa and Velcade (btz) have cantly more sensitive to combination therapy rather than Mapa been used in a clinical trial with no additional effect of the anti- alone (Fig. 1); however, the Mapa-resistant cell lines (NCI- body above that of Velcade.27 This combination however may H929 and JJN-03) were also resistant to LDBCMapa therapy. not be optimal at the current bortezomib doses used clinically. Interestingly, LP-1 was only partially sensitive to combination btz has numerous immunosuppressive effects on the human therapy (30% apoptosis); there was no statistical significance in immune system,28-30 contributing to a demonstrable increase in the extent of LP-1 apoptosis induction by Mapa alone and the incidence of Varicella infections in btz-treated MM LBMCM combination therapy. patients.31 combination therapies using We next asked whether sequential treatment (LDB followed standard dose btz, may therefore be limited in their ability to pro- by Mapa) would render the myeloma cells more sensitive to mote immune responses by the immunosuppressive effects of btz. LDBCMapa-induced apoptosis. Pre-treatment of human mye- In this study, we addressed this problem by adjusting the dose loma cell lines (HMCL) with LDB induced increased TRAIL-R1 of btz to below that able to induce single agent myeloma cell apo- expression (compared to untreated cells) in LP-1 and OPM-2 ptosis and then explored whether myeloma cells pre-treated with cells (Fig. 1B). To explore whether this LDB pre-treatment LDB potentiated Mapa-induced apoptosis. We also explored could significantly improve LDBCMapa-induced myeloma cell whether antibody-mediated tumor cell death induction also pro- apoptosis, three HMCL’s (LP-1, NCI-H929 and JJN-03) were moted antitumor immune responses and the mechanisms tested as they previously showed no increase in apoptosis in required to do so. Our results indicate that in an NK cell-depen- response to the LDBCMapa combination (compared to Mapa dent manner, the combination of LDBCMapa therapy induces alone). Following pre-treatment with LDB, LP-1 cells were sig- myeloma cell immunogenic death and promotes potent anti- nificantly more sensitive to LDBCMapa combination therapy myeloma cytotoxic cell immunity. than when treated with combination therapy alone (Fig. 1C). In contrast, JJN-03 and NCI-H929 cells were resistant to the two phase ‘LDB’ followed by combination therapy (data not shown). Results Taken together, this data showed the combination LDBCMapa treatment effectively induced myeloma cell apopto- TRAIL receptor-1 expressing human multiple myeloma cell sis in vitro and that sequential treatment of myeloma cell lines lines are sensitive to Mapa plus low dose bortezomib with LDB followed by Mapa also shows promising antitumor Six MM cell lines, when tested for sensitivity to titrated doses activity. of btz (Fig. S1), revealed two features. First, MM cell lines were sensitive to btz from a 5 nM dose and above. Secondly, the sub- Human dendritic cells treated with low dose bortezomib optimal apoptosis-inducing dose was established for each cell line function normally and was 1 nM btz for RPMI8226, U266 and NCIH929; Human DC viability and function is compromised in vitro by 2.5 nM btz for OPM-2, JJN-03 and 5 nM for LP-1. This btz btz,28,29 this occurs from a 10 nM dose upwards (data not dose was used in subsequent studies to stress the MM cell with- shown). Furthermore, a prior study using a xenotransplant model out inducing apoptosis, and was termed “LDB”. MM cell line of MM32 showed proteasome inhibition occurs in the peripheral expression of TRAIL-R1 was shown to be variable; thus, LP-1, tissues and lymphoid organs within 1 h of dosing. Lastly, the btz RPMI8226, NCI-H929, U266 and OPM-2 all express TRAIL- dose used in clinical practice (1.3 mg/m2/dose i.v.) results in pro- R1 (Fig. S2A) constitutively. In contrast, JJN-03 was consis- teasome inhibitor activity in the peripheral blood (PB), based on tently negative for TRAIL-R1. Subsequently, all cell lines were animal studies and antitumor activity, and this is likely the case treated with titrated doses of Mapa, and apoptosis assessed at in peripheral tissues too. We examined whether using lower dose 48 h. The TRAIL-R1 expressing cell lines (OPM-2, U266 and btz combined with Mapa would retain anti-myeloma immune LP-1) showed 60%, 30% and 25% apoptosis in response to activity, including DC function. To do this, we performed a 10 mg/mL Mapa respectively (Fig. S2B). RPMI8226 is exqui- series of studies examining DC function in increasingly stringent sitely sensitive to Mapa and undergoes apoptosis (90–100)% in drug conditions. Initially, inhibition of proteasome chymotryp- response to 0.14 mg/mL Mapa (Fig. S2C). Two cell lines (NCI- sin (Ch)-like activity was assessed on LDB, Mapa or H929 and JJN-03) were resistant to Mapa therapy in the dose LDBCMapa-treated monocyte-derived dendritic cells (MoDCs) range 0–10 mg/mL (Fig. S2B). Whilst TRAIL-R1 expression (Fig. S3). This experiment showed that LDB and LDBCMapa was required for the Mapa response, TRAIL-R1 expression level inhibited proteasome Ch-like activity by 10%, whereas Mapa did not correlate with the extent of Mapa-induced apoptosis alone had no effect.

e1038011-2 OncoImmunology Volume 4 Issue 9 We then performed complemen- tary studies to examine MoDC phagocytosis of apoptotic myeloma cells. First, live video microscopy was used to examine the kinetics and morphology of apoptotic mye- loma (apo-MM) phagocytosis by DCs (Fig. 2A). This study showed that Apo-MM were phagocytosed by DCs as one large body within 20 min of co-culture, and that by 40 min the Apo-MM phagosome had matured (drop in pH reflected by pHrodobright fluorescence). The pHrodobright Apo-MM remained in a mature phagosome for a further 1.5 h. Second, FACS was used to examine whether there was a quanti- tative difference in DC phagocytosis of Apo-MM depending on the drug used to induce MM cell apoptosis (Fig. 2B). Thus, DCs phagocytosed untreated RPMI8226 cells (15.69 § 2.5)%; in contrast, a significant increase in MM phagocytosis was observed when the RPMI8226 cells were pre-treated with 1 nM Btz alone, Mapa alone or LDBCMapa (Fig. 2B). Interestingly, pre-treat- ment with Mapa alone or LDBCMapa induced a significantly higher level of DC phagocytosis compared to LDB pre-treatment alone, indicating that Mapa-medi- ated the dominant role in the increased DC phagocytosis observed with LDBCMapa treated MM cells. Equivalent results were observed using treated U266 cells in the DC phagocytosis assay (Fig. S4). Next, the effect of combination drug treatment on lipopolysaccha- ride (LPS)-induced DC maturation was examined. Thus, when MoDCs were stimulated with LPS, there Figure 1. Combination low dose bortezomib plus mapatumumab induce human myeloma cell apoptosis. was no significant difference in (A) Six human myeloma cell lines were cultured for 48 h in triplicate wells in the previously determined sub- DC maturation (CD80, CD86, optimal btz dose, or increasing doses of Mapa (0.01–50 mg/mL) alone, or the combination of LDBCM. HMCL CD83, HLA-ABC and HLADR apoptosis was measured by annexin V binding and is presented as the mean § SE of three separate experi- fi p D < expression levels) between ments, * designates a statistical signi cant difference (where 0.05) in HMCL apoptosis between Mapa alone and the combination of Mapa and low dose bortezomib. (B) HMCL were either untreated, or treated untreated DCs or DCs treated for 24 h with LDB. TRAIL-R1 expression was determined by FACS staining. Data is shown as histogram over- with LDB, Mapa or LDBCMapa lays for each HMCL, and is representative of triplicate wells from three separate experiments showing consti- (Fig. 2C). There was also no sig- tutive TRAIL-R1 (thick black line), TRAIL-R1 after 24 h LDB treatment (dotted line), isotype control (shaded nificant difference in the level of light gray) or secondary only control (dashed line). In brackets are MFI values for HMCL TRAIL-R1 expression IL-12p70 or IL-4 induced by after 24 h LDB treatment (Pre-btz) or untreated (U/T). In C, LP-1 cells were either untreated (black) or pre- treated with 5 nM Btz (white) for 24 h prior to the addition of Mapa (0.01–50 mg/mL) for 48 h. LP-1 apopto- LPS in the presence or absence sis was measured by annexin V binding and is presented as the mean § SE of three separate experiments, * of drug co-treatment (Fig. 2D). designates a statistical significant difference (student t-test where p D <0.05) in LP-1 apoptosis between untreated versus LDB pre-treated cells. www.tandfonline.com OncoImmunology e1038011-3 Figure 2. For figure legend, see next page.

e1038011-4 OncoImmunology Volume 4 Issue 9 Figure 3. Combination LDBCM induced increased effector cell killing of human myeloma cells. HLA-A2*01 MoDCs were pulsed with apoptotic U266 cells (induced by LDBCM treatment) in the presence or absence of LDBCM. MM pulsed-DCs were then cultured with autologous PBMCs and the responder T cells expanded for 21 d in vitro (see Methods for details). The expansion of MM-specific effector cells was then assessed by CTL assay using Cr-labeled U266 cells as targets. Data presented is the percentage lysis (mean § SE) of targets for three separate experiments on four HLA-A2*01 donors. Data series include four separate test conditions in the in vitro culture. These include DC pulsed with untreated or treated U266, DC pulsed with treated U266 in the presence of btzCMapa, DC pulsed with treated U266 and then matured with LPS. Additional controls included ‘unloaded DC’ used at the priming phase in the culture (but U266 as targets in the killing assay), or control targets (autologous PBMC control) to show specificity of the AMCL response. A statisti- cal (student t-test) difference in U266 lysis between the test and control combinations was shown, where * p < 0.05, and ** p < 0.01.

Despite the immunosuppressive nature of apoptotic d. The MM killing assay showed untreated or LDBCMapa- myeloma cells, myeloma-pulsed dendritic cells induce an treated U266 suppressed the ability of DCs to induce expansion anti-myeloma immune response of an anti-MM cytotoxic response (Fig. 3). In contrast, a signifi- Whilst LDB treatment did not inhibit LPS-induced MoDC cant expansion of MM-specific CTLs occurred when Apo-U266- maturation, phagocytosis of apoptosis MM cells was associated pulsed DCs were also treated with LDBCMapa during the prim- with impairment of DC maturation. Prior co-culture of DCs ing phase of the culture (Fig. 3). The level of CTL expansion with LDBCMapa-treated U266 or RPMI8226 cells significantly was similar to that observed when Apo-U266-pulsed DCs were inhibited LPS-induced DC maturation (Fig. S5), as assessed by activated with LPS (Fig. 3). This cytotoxic response was specific CD80, CD83 and CD86 expression. LPS-induced MoDC to the HMCL, since autologous PBMCs were not lysed by effec- CD80 and CD86 expression was more significantly inhibited by tor cells (Fig. 3). In addition, the AMCL expansion required Apo-RPMI8226 (p < 0.01) than by Apo-U266 (p < 0.05). DCs pulsed with myeloma antigen, as the ‘no antigen’ DC con- Using this information, a modified in vitro expansion culture trol did not induce AMCL effectors (Fig. 3). of anti-MM lymphocytes was devised using either no myeloma cells, untreated U266, or LDBCMapa treated U266 as an anti- LDBCMapa combination therapy does not exert the gen source. Furthermore, U266-pulsed DCs were then either immune-modulating effect via a direct effect on dendritic cells treated with LDBCMapa or LPS. Autologous peripheral blood or by differential induction of TRAIL-R1 on immune subsets mononuclear cells (PBMCs) were added to the MM-pulsed To explore the mechanism/s whereby LDBCMapa expanded MoDCs for 7 d, and anti-myeloma cytotoxic lymphocyte AMCLs, we first examined the direct effect of LDBCMapa on (AMCL) expansion performed (as per methods) for a further 14 MoDC function. We know that MoDC effectively phagocytose

Figure 2 (See previous page). Combination LDBCM treatment of human myeloma cells induced increased MoDC cell phagocytosis, but does not affect MoDC response to the TLR ligand LPS. HMCL (U266 and RPMI8226) were labeled with the pH sensitive dye pHrodo, pre-treated with LD btz and Mapa for 48 h, then co-cultured with MoDCs (as per Methods). The kinetics of MoDC phagocytosis of apoptotic HMCLs was monitored by live video microscopy (A), then quantitated by flow cytometry (B). The effect of LDBCM combination or single drug treatment on MoDC responses to LPS were assessed in C and D.InC, changes in MoDC expression of CD80, CD86, CD83, HLA-ABC and HLADR and CD83 were investigated. Data shown is representative histo- gram overlays, and collated mean fluorescence intensity (MFI) data from triplicate wells of a single experiment. In D, MoDC secretion of IL-12p70 and IL- 4 is shown. Data presented is either representative data from three separate experiments (A) and (C). Quantitative data in B and D is the mean § SE for three separate experiments, p values (student t-test) are provided in B for test conditions that are significantly different to the control, and in D **** rep- resents p < 0.0001.

www.tandfonline.com OncoImmunology e1038011-5 Figure 4. For figure legend, see next page.

e1038011-6 OncoImmunology Volume 4 Issue 9 LDBCMapa treated MM cells; how- ever, does this facilitate MoDC matura- tion/licensing in the absence of a toll- like receptor (TLR)-mediated signal? To address this question, we assessed whether MoDC maturation was affected by co-treatment with LDB- Mapa in the presence or absence of U266 MM cells (as depicted in Fig. 4A). We examined MoDC CD80, CD83, CD40 and HLADR expression levels at 4 and 48 h after initiation of LDBCMapa treatment. In the absence of LPS, we showed no change in MoDC maturation kinetics in the con- text of LDBCMapa alone (Fig. 4B), whether the MoDCs were also pulsed with U266 or not. In the presence of LPS, MoDCs up-regulated CD80, CD40, CD83 and HLADR by 48 h, this response (CD80, CD83 and CD40) was significantly decreased Figure 5. NK cells are required at the priming phase for efficient effector cell killing of human mye- when the MoDCs were first pulsed with loma cells. A myeloma-specific effector cell culture was established as for Fig. 3. Thus, MM-pulsed LDBCMapa-treated U266 (Fig. 4B). MoDCs were co-cultured with autologous PBMCs for 7 d. In addition, monocytes or NK cells were In addition, we explored whether the depleted from the priming phase of the effector cell culture. MM-specific effector cells were expanded LDBCMapa co-treatment could with re-stimulation and expansion steps up to day 21. Harvested effector cells were used in a killing assay with Cr-labeled U266 as targets (E:T D 25:1). Results presented are percentage lysis (mean § SE), directly alter MoDC-secreted cytokine pooled data from three separate experiments. Where a statistical difference (student t-test) in U266 levels. To address this, drug treated killing was observed ** p D <0.005, ***p D <0.0005, ****p D <0.0001. MoDC culture supernatant was col- lected at early and late timepoints (0, 4,8 and 24 h) and a cytokine bead array performed for GM-CSF, IL-4, IL-6, IL-12p70, IL-1b, IL-6, IL- 48 h time course (Fig. S6B). In addition, lymphocyte subset fre- 10 and sCD40L levels (Fig. 4C). We showed the LPS control quencies and their TRAIL-R1 expression were also assessed dur- MoDCs secreted increased levels of all cytokines except IL-4. In ing the anti-myeloma expansion culture, using the expansion the absence of LPS, apo-U266 pulsed MoDCs secreted higher culture conditions described in Fig. S7. Data showed the per- levels of IL-4 than non-pulsed MoDCs. This was true whether centage of different lymphocyte subsets changed during the the MoDCs were also treated with LDB, Mapa or LDBCMapa expansion culture, although this was not specific to the (Fig. 4C). There was no data to indicate that LDBCMapa treat- LDBCMapa treatment group. For example, NK cell percent- ment of MoDCs induced increased IL-12p70 or Il-1b secretion. age increased at one week after 1st restimulation; however, An alternate potential mechanism for the LDBCMapa-treat- this was the case for all treatment groups. The percentage of C ment effect on CTL expansion was a differential effect on the TRAIL-R1 Tregs increased in all culture conditions across effector:suppressor ratio of T cells and their expression of the timecourse; in contrast, NK and B cells had a specific TRAIL-R1, the target for Mapa. To address the latter, human TRAIL-R1 increase in the Apo-U266 MoDCCLPS control. PBMCs were directly treated with LDBCMapa and TRAIL-R1 Taken together, in the LDBCMapa versus other treatment assessed at 4 and 48 hours (depicted in Fig. S6A). TRAIL-R1 groups,therewasnodatatoindicatearelativeincreasein C C C expression on regulatory CD4 T cells (Treg), CD4 or CD8 Teff or NK cell:Treg ratio, or a specific increased expression T cells was not affected by LDBCMapa treatment during this of TRAIL-R1 on these subsets.

Figure 4 (See previous page). Combination LDBCMapa treatment does not directly alter MoDC maturation or cytokine secretion. MoDCs were cultured with U266 myeloma cells and the effect of subsequent LDBCMapa treatment on MoDC maturation compared to MoDCs activated with LPS. MoDCs were harvested at 4 and 24 h post drug or LPS treatment (as depicted in A) and assessed for the changes in the MoDC maturation markers CD80, CD40, CD83 and HLADR (B). Data show is protein expression assessed by FACS in mean fluorescence intensity (MFI). This is pooled data of therapy responses from three separate normal donor MoDC cultures. A statistically significant difference between test and control groups is represented by * (p < 0.05), **(p < 0.01), ***(p < 0.001). In addition, in C culture supernatant was collected from the co-cultures at 4, 8 and 24 h and cytokine levels (IL-12p70, IL-8, IL-4, GM-CSF, IL-1b, IL-6, IL-10 and sCD40 ligand) assessed by Luminex assay. In these co-culture conditions, ‘tr’ refers to cells treated with LDBCMapa. Data is from triplicate wells of a single experiment, representative of two experiments.

www.tandfonline.com OncoImmunology e1038011-7 Figure 6. Myeloma induced activation of NK cells is increased by Mapatumumab pre-treatment. U266 myeloma cells were either untreated, or treated with LDB, Mapa or LDBCMapa for 48 h (as per methods) and the effect of drug treatment on myeloma cell immunogenicity assessed by FACS for expres- sion of CD80, CD86, CD83, HLA-ABC, HLA-DR (A). The effect of drug treatment on myeloma cell NK cell activating ligand expression was also investigated (B). NK activating ligands included the NKG2D ligands MIC-A, MIC-B, ULBP-1, ULBP-2,5,6 and the DNAM-1 ligands CD155 and CD112. Data is presented as representative histogram overlays for the isotype control (filled light gray), untreated (filled dark gray), Mapa (black dotted line), LDB (black line), or LDBCMapa (black dashed line) treated U266 cells. In addition, in B a statistical comparison was made between treatment groups for NK cell activating ligand expression. In C at 48 h, the treated U266 cells were also co-cultured with HLA-A2+ PBLs. Cells were harvested at 4 (A)or24(B) h and the effect of drug treated myeloma cells on NK cell and T cell subset activation assessed by CD69 and CD25 expression. Data is presented for triplicate wells in a single experiment, representative of two separate experiments.

e1038011-8 OncoImmunology Volume 4 Issue 9 Figure 7. For figure legend, see next page.

www.tandfonline.com OncoImmunology e1038011-9 NK cells play a key role in expansion of anti-myeloma U266 cells were pre-treated with LDBCMapa for 48 h, MoDCs cytotoxic lymphocytes were pulsed with the drug-treated U266 and then co-cultured Having excluded an immediate direct effect of LDBCMapa with autologous PBLs. Co-cultured cells were assessed for treatment on DCs, we next explored whether the LDBCMapa- changes in the activation markers CD25 and CD69 at 4 and induced AMCL expansion occurred via an indirect effect at the 24 h. At 24 h, MoDCs induced an increased percentage of acti- C C C C priming phase of the culture (Fig. 5). We postulated that FcgR vated (CD69 CD25– and CD69 CD25 ) NK, CD4 and C expressing NK cells or monocytes could be activated via Mapa CD8 T cells when the MoDCs were pulsed with untreated and change the subsequent expansion of AMCL by an indirect U266 (CLPS) or pulsed with LDBCMapa treated U266 mechanism. Thus, when NK cells (and not monocytes) were (Fig. 7A, B). Analysis of NK subset responses (Fig. 7B) showed C depleted at the start of the culture from the autologous PBMCs, an increased percentage of CD25–CD69 cells within both the C C ¡ anti-myeloma cytotoxic function was significantly decreased CD16 CD56 and CD56hiCD16 NK cells when the MoDCs (Fig. 5). Using the 51Cr-release assay to test anti-myeloma killing were pulsed with U266 cells (in the presence or absence of C capacity of the ensuing expanded cells, we showed that both LDBCMapa). In the same culture conditions, both CD4 and C C ¡ T cells (U266 killing in the presence of cold K562 targets) and CD8 T cells showed an increased percentage of CD25 CD69 NK cells participated in the myeloma target killing (Fig. 5). cells. Having revealed that U266 pulsed MoDCs activated NK and Mapatumumab plays a key role in NK cell direct activation T cell subsets within the first 24 h of the co-culture with autolo- by LDBCMapa-treated myeloma cells gous PBLs, we next explored whether these activated cells prolif- To explore how NK cells may be affected during the AMCL erated over 7 d of culture. To do this, we labeled the autologous expansion culture-priming phase, we examined potential direct PBLs with Cell Trace Violet (CTV) and examined NK and T molecular interactions between the drug-treated U266 and subset proliferation by CTV dilution assay plus their activation C immune system cells, with a focus on NK cells and their activa- status. This study revealed that all NK cell subsets and CD8 T C tion. To do this, we first examined whether drug treatment of cells, (but not CD4 T cells) undergo increased proliferation U266 cells affects their immunogenicity or expression of NK- when co-cultured with MoDCs pulsed with U266, either after activating ligands. We showed that U266 cell immunogenicity LDBCMapa pre-treatment or with LPS activation (Fig. 8A). In C (CD80, CD83, CD86, HLA-ABC and HLA-DR levels) was not the same co-cultures, all NK cell subsets and CD8 T cells had C affected by single LDB, Mapa or LDBCMapa treatment an increased percentage of CD25 cells, this correlated with (Fig. 6A). Furthermore, we observed that U266 cells constitu- increased proliferation in the context of U266-pulsed MoDCs C tively expressed the NKG2D ligand MIC-A and low levels of the (Fig. 8A). Interestingly, CD4 T cells displayed a marked C DNAM-1 ligand CD112; expression of these NK cell-activating increase in the percentage of CD25 cells when PBLs were cul- ligands was not altered by single LDB, Mapa or combination tured alone, this decreased when the PBLs were co-cultured with LDBCMapa treatment (Fig. 6B). When the drug-treated U266 U266 pulsed MoDCs. In addition, day 7 expanded cells from cells were then co-cultured with PBLs, NK cells were activated at the co-culture were also re-stimulated with U266 cells to assess both the 4 and 24 h time points (Fig. 6C). Higher levels of NK effector function status at this time point (Fig. 8A). To assess the activation were observed in wells where PBLs were co-cultured functional status of these activated proliferating NK and T cells, with Mapa- or LDBCMapa-treated U266 cells, suggesting that we co-cultured the cells with either LDBCMapa-treated or Mapa plays the key role in mediating the LDBCMapa mediated untreated U266 cells for a 4 h period and assessed whether they NK cell activation (Fig. 6C). We also observed a lower but sig- degranulated (CD107a), and/or secreted cytokines. The nificant increase in NK cell activation in the context of untreated LDBCMapa treated or untreated myeloma cells induced NK cell C C U266 cells (vs. PBL alone control), this is likely due to recogni- subset and CD8 T cell degranulation (CD107a ), a subset of tion of U266 NK activating ligands by the responding NK cell these cells also produced IFNg and TNF-a (Fig. 8B). This C (ie.NKG2D/MIC-A and DNAM-1/CD112) (Fig. 6C). shows the U266-pulsed MoDCs induce effector NK and CD8 T cells; in these culture conditions, there was no apparent differ- C Myeloma-pulsed DCs induce NK and CD8 T cell ence in responses by these cells when re-stimulated in the context activation and proliferation of untreated or LDBCMapa-treated U266. In the final phase of this study, we explored whether the In summary, LDBCMapa treatment of myeloma-pulsed DCs MoDCs (pulsed with drug-treated U266) induced activation of induces a powerful CTL response, this does not occur via a direct NK and T cell subsets within 24 h of co-culture. To do this, effect on DCs, effector T cells or Tregs. Rather Mapa has two

Figure 7 (See previous page). Myeloma-pulsed DCs induce increased activation of autologous NK, CD4C and CD8C T cells. Monocyte-derived DCs were pulsed with untreated or drug (Mapa, LDB or LDBCMapa) treated U266 cells (as per methods) for 2 h, then co-cultured with autologous normal donor (n D 2) PBLs for a further 24 h. Cells were collected at 4 and 24 h, and activation of NK cells and T cell subsets assessed by CD69 and CD25 expression. Data presented in A are representative dot plots of NK and T cell activation in cultures from a single normal donor. Shown are PBL alone, PBLCDC, PBLCU266 pulsed MoDC (where U266 were pre-treated with LDBCMapa), and PBLCU266 pulsed MoDC in the context of 1 mg/mL LPS. Collated data in B is from duplicate co-culture wells (single normal donor representative of two normal donor experiments) and shows the percentage of CD25¡CD69C, CD25CCD69C and CD25CCD69¡ cells in NK cell subsets, CD4C and CD8C T cells.

e1038011-10 OncoImmunology Volume 4 Issue 9 Figure 8. For figure legend, see next page.

www.tandfonline.com OncoImmunology e1038011-11 distinct modes of action which co-opt the immune system indi- IL-12, IL-18, IL-15 and type I interferon and contact-dependent rectly. First, Mapa induces NK cell activation within 24 h of signals (NKG2DL-NKG2D, GITRL-GITR, LFA1-ICAM1),36 contact with LDBCMapa treated myeloma cells. Second, Mapa which can have a dramatic effect on the functional capacity of promotes DC phagocytosis of LDBCMapa treated myeloma the DC and ensuing T and NK cell responses. We did not see cells. We also know there is an ensuing antigen-specific response any effect of LDBCMapa treatment on cytokine secretion by C by both NK and CD8 T cells within 24 h and proliferation by MoDCs in the context of treated myeloma cells. However, this 7 d to produce fully functional effector cells. does not exclude the possibility that the cross-talk occurs between NK cells and DCs at a later time point in our culture system. It is likely the NK cells are initially activated via interactions between Discussion activating receptors and activating ligands expressed on myeloma cells. In addition, Mapa-bound to either apoptotic or viable mye- Until recently, it has proven difficult to develop effective loma cells will be bound by NK cell FcgRIII. The net effect of immunotherapies against MM. This is in part due to the absence these interactions is increased NK cell activation, degranulation of effective myeloma targeting antibodies. Recently, two mono- and production of cytokines,37 including IFNg, which has a DC clonal antibodies, Elotuzumab (anti-SLAMF7) and Daratumu- licensing effect.38 This DC licensing is likely to have occurred mab (anti-CD38), have shown pre-clinical and early clinical trial following cross-talk with activated NK cells present within the success. Whilst both clearly rely on NK cell function to induce culture.39,40 Whilst FcgR-mediated phagocytosis of antigen is maximal plasma cell apoptosis, little is known about their down- known to be an efficient pathway by which to load DCs with stream ability to induce immunogenic cell death and prime cyto- antigen41,42 induction of an antigen-specific T cell response toxic cellular immunity. We show that the agonistic TRAIL-R1 requires a further licensing signal for the DCs. In our study, this antibody Mapa may also act as a myeloma-targeting antibody. is efficiently achieved by Mapa-activated NK cells providing a We revealed that Mapa has at least two distinct modes of anti- DC licensing signal. myeloma activity. First, Mapa induces myeloma cell apoptosis Taken together, our study showed Mapa is a novel anti-mye- directly, which is increased via pre- or concurrent treatment with loma antibody that has multiple effector arms. The rational com- LDB. These results compare favorably to studies where Mapa bination with LDB enhanced optimal myeloma cell apoptosis was used in vitro to induce HMCL and primary myeloma cell leading to effective immunogenic cell death mediated by pre- death.25,33 served DC function and NK cell mediated licensing. Our study Secondly, we show that Mapa induced plasma cell killing eli- provides a strong rationale for the application of LDBCMapa in cited a powerful anti-myeloma cytotoxic response. Critically, this clinical trials of MM immunotherapy. anti-myeloma CTL expansion occurred optimally when both Mapa and NK cells were present during the priming phase. Indeed, compared to LDB treatment, Mapa-treated myeloma Methods and Materials cells significantly increased NK cell activation and led to the max- imal uptake of apoptotic cells by DCs, which in turn resulted in Cell lines and materials C reproducible expansion and activation of cytotoxic CD4 and Human myeloma cell lines RPMI8226, U266 and NCI- C CD8 T cells. H929 cell lines were obtained from American Type Cell Collec- Prior studies have shown that apoptotic myeloma cells inhibit tion (ATCC); LP-1, OPM-2, and JJN-03 cell lines were from LPS-induced DC maturation due to a combination of IL-10, DSMZ (Braunschweig, Germany). Cell lines were cultured in TGF-b and VEGF inducing activated phospho-STAT-3.34,35 RPMI1640 supplemented with 10% heat-inactivated fetal bovine Our study confirms this observation. However, despite this, serum and 2 mM L-glutamine. btz was from Millenium Pharma- when autologous PBMCs were primed in a co-culture system ceuticals, MA; Mapa from Human Genome Sciences Inc., NJ; containing LDBCMapa, myeloma targets, PBMC and DCs, an Annexin V-FITC, 7-AAD, anti-mouse Ig-PE, the monoclonal expanded population of myeloma-specific cytotoxic cells was antibodies CD11c-PECy7, CD83-APC and HLADR-FITC consistently generated. The importance of NK cells during the were from BD Biosciences, CA and the IL-12p70 EIA kit from co-culture priming phase suggests cross-talk between NK cells BD PharMingen, CA. pHrodoTM (Invitrogen, Carlsbad, CA); and the Apo-MM pulsed DCs. It is known that NK-DC cross lipopolysaccharide (from E.coli, SIGMA ALDRICH, St Louis, talk involves a number of mechanisms including soluble factors Mo).

Figure 8 (See previous page). Myeloma-pulsed DCs induce increased proliferation of NK cells, CD4C and CD8C T cells. Monocyte-derived DCs were pulsed with untreated or drug (Mapa, LDB or LDBCMapa) treated U266 cells (as per methods) for 2 h, then co-cultured with autologous normal donor (n D 2) PBLs (pre-labeled with Cell trace Violet, CTV) for 7 d. Cells were harvested, labeled with conjugated antibodies and analyzed on the FACS. In A, the proliferation and activation status of NK and T cell subsets was shown by gating first on lineage markers (NK subsets: CD56hiCD16¡, CD16-CD56lo, CD56loCD16C, and T cells CD3CCD4Cand CD3CCD8C) followed by CTV dilution (left panels) or CD69,CD25 expression (right panels). Cell proliferation data is shown as individual histogram overlays with the PBL alone control (filled light gray) with the test groups (thick black line) either PBLCU266-pulsed MoDC or PBLCU266 pulsed MoDC (where U266 were pre-treated with LDBCMapa). In B day 7 cells were recovered from the co-culture and stimulated by co-culture for 4 h, cells were stained for activation markers (CD25,CD69), degranulation (CD107a), and cytokines (IFNg and TNF-a). Data is presented as dot plots showing CD107a expression and TNF-a (left panel) or IFNg (right panel) production by NK or T cell subsets.

e1038011-12 OncoImmunology Volume 4 Issue 9 Myeloma therapy experiments DCs were then added to wells and positions were selected to Myeloma cells (2 £ 106/mL) were treated with mono- or maximise the wells in the field of view with DCs clearly visible. combination therapy for 48 h in 24 well plates. Initially, drugs The tumor target cells were added (2 £ 105) just prior to were used as monotherapy including btz (0–10 nM) or Mapa the image sequence starting. Images were taken at each position (0–10 mg/mL) and apoptosis induction assessed. To do this, every 4–5mins for 3–4 h. The objective of the microscope was after 48 h treatment, cells were harvested, washed once in 20£. The images were collated and analyzed using Metamorph Dulbecco’s PBS and then stained with Annexin V-FITC and Software (MDS Analytical Technologies, Downington USA), 7AAD for 10 min at room temperature. Stained cells were then which allows single frame images to be viewed as a multi-frame analyzed on the BD LSRII flow cytometer. For combination video. therapy, myeloma cell lines were treated with the highest dose of btz that did not induce apoptosis (termed ‘low dose’, this dose LPS-induced DC responses was established from the monotherapy titration) combined with MoDCs were cultured with 1 mg/mL LPS for 48 h at 37Cin titrated amounts of Mapa (from 0–10 mg/mL) for 48 h prior to the presence or absence of 1 nM btz. MoDCs were harvested and evaluation of myeloma cell apoptosis. stained with antibodies to CD11c, CD83 and HLA-DR, cells were analyzed on the BDLSRII flow cytometer to examine upre- Combination Mapa and low dose bortezomib therapy do not gulation of CD83 and HLA-DR as an indicator of LPS-induced inhibit dendritic cell function MoDC maturation. In addition, culture supernatant was col- MoDCs were established from fresh PB monocytes as lected at 48 h of LPS treatment and an IL-12p70 assay per- described previously.43,44 Briefly, purified monocytes were cul- formed (as per manufacturer’s instructions). tured for 5 d in 10 ng/mL IL-4 and 20 ng/mL GM-CSF (Pepro- tech, NJ), the media was renewed at day 3. On day 5, non- DC proteasome activity assay (chymotrypsin-like functional adherent cells were removed by gentle aspiration from the culture subunit) flask and the purity of immature MoDCs checked by FACS MoDCs were cultured (as described above), proteasomes were ¡ using the phenotype CD11chi/CD14 /CD11bint. extracted from the DCs and treated with 1 nM btz alone, 1 mg/ mL Mapa alone or a combination of both drugs for 4 h at 37C. DC phagocytosis of treated myeloma cells Treated proteasomes were pelleted and then a Ch-like protea- MoDCs were harvested from the above cultures and used for some assay performed (Millipore), as per manufacturer’s instruc- myeloma cell phagocytosis assays. To induce myeloma cell apo- tions, and46,47 using Suc-Leu-Leu-Val-Tyr-MCA as the 20S ptosis, RPMI8226 were either g-irradiated (30 Gy), or treated proteasome substrate. with 1 nM btz alone, 0.9 mg Mapa alone or a combination of both drugs for 48 h. Treated myeloma cells were stained with Expansion of myeloma-specific cytotoxic lymphocytes 0.5 mg/mL pHrodo, washed with PBS twice before co-culture in vitro and detection of cytotoxic function with MoDCs (1:2 myeloma:DC ratio) for 4 h at 37C. A nega- U266 myeloma cells were either untreated, or treated with tive control co-culture was also performed at 4C. Harvested cells LDBCMapa for 48 h to induce apoptosis (Apo-U266). U266 or were then stained with anti-CD11c-PECy7 and Fluorogold Apo-U266 cells were washed twice in RPMI1640 containing (Invitrogen); stained cells were analyzed on the BD LSRII flow 10% fetal bovine serum, complete medium (CM). MoDCs cytometer, 5000 CD11chi events were collected and the fre- (HLA-A2*01 normal donor) were pulsed with either untreated quency of CD11chi/pHrodohi collected. In other experiments, or Apo-U266 cells for 4 h in the presence or absence of the MoDC myeloma cell phagocytosis assay was performed in LDBCMapa. Subsequently, the U266-pulsed MoDCs were co- the presence of 1 nM btz. pHrodoTM is a pH sensitive dye and cultured with autologous PBMCs for 7 d in CM containing undergoes a quantum yield in red fluorescence when stained apo- 20 U/mL IL-2, 10 ng/mL of IL-7 and IL-15 (priming phase). ptotic bodies are processed to the low pH environment of an On days 7 and 14, cells were harvested and re-stimulated with endosome. Thus, cells that are CD11chi/pHrodohi are MoDCs autologous myeloma-pulsed MoDCs, expansion of CTL during with endocytosed myeloma cells and CD11chi/pHrodolo are these time points occurred in CM supplemented with 25 U IL- likely MoDCs with adherent myeloma apoptotic bodies. To ver- 2. On day 21, cells were harvested and their anti-myeloma activ- ify that the FACS data did indeed represent MoDC phagocytosis ity assessed by a chromium-release assay. Controls for the CTL of treated myeloma cells we examined an aliquot of the same cells expansion culture include unpulsed MoDCs, or U266-pulsed by fluorescent live video microscopy. To do this, apoMM-pulsed MoDCs that were activated with 1 mg/mL LPS. Chromium- MoDCs (1 £ 105) were added to optimally manufactured grids release assay was performed as described previously.9 Briefly, [125 mm (l) £ 125 mm (w) £ 60 mm (h)], which are individual chromium-labeled U266 cells were cultured (in triplicate wells) paddocks designed to contain the cells within the microscope with effector cells from the CTL expansion culture at an effector- field of view (as described previously45 The grids were then target ratio (E:T) from 6.25 to 50:1. After 4 h, culture superna- placed in imaging chamber slides (Ibidi, DKSH, Martinsried, tant was collected from each well and the amount of Cr released Germany). Images were taken on the Leica SP5 confocal micro- by U266 cells measured on a g-counter (Wallac Wizard 1470, scope (Leica, Wetzlar, Germany). Voltage gain, offset and focus Perkin Elmer, MA, USA). The amount of U266 cell death (per- were set on compensation wells before the first image was taken. centage lysis) was compared to total U266 Cr-release wells

www.tandfonline.com OncoImmunology e1038011-13 (U266 plus 2M HCl). Thus, percentage lysis was calculated using analyzed by FACS, and co-culture induced activation of NK or the formula (mean cpm – spontaneous cpm)/total cpm. To assess T cell subsets assessed following a devised gating strategy depicted the contribution of different effector cells (NK versus T cell) to in Fig. S8. the killing of U266 cells, a ‘cold target assay’ was performed. To do this, the Cr-release assay was performed as above, except that Detection of NK or T cell subset activation following co- in addition to the Cr-labeled U266 cells, equal numbers of unla- culture with myeloma-pulsed MoDCs beled K562 cells (the cold target) were included. To assess the rel- Day 5 MoDCs were pulsed with drug-treated U266 cells at a ative importance of NK cells or monocytes at the priming phase 1:1 ratio as in the protocol for CTL expansion culture (see ¡ C of the CTL expansion culture, NK cells (CD3 /CD56 )or above). After 4 h, cells were washed twice and left overnight. C monocytes (CD14 ) were removed from the autologous PBMCs Recovered autologous PBLs were washed, counted and added to by FACS-sorting, purity was >95%. The NK- or monocyte- the pulsed MoDCs at a 5:1 ratio. Co-cultured cells were recov- depleted PBMCs were then co-cultured with MM-pulsed ered 4 and 24 h later. Four hours prior to each timepoint, Golgi MoDCs as above. stop and CD107a-PeCy5 (BD) antibody were added to the appropriate wells for intracellular staining. Cells were washed Direct effect of LDB§Mapa treatment on MoDC and stained for NK, CD4 and CD8 T cell markers (CD3- maturation and cytokine secretion BV711, CD4-BV605 (both Biolegend), CD56-PECy7, CD8- Day five MoDCs were divided into two groups, one was PerCPCy5.5, CD14/CD19-APCH7, CD16-PE (all BD Bio- unpulsed and the other pulsed with LDBCMapa treated U266 sciences). Cells were then fixed and permeabilized (BD Cytofix/ myeloma cells for 4 h. Both MoDC groups were then each fur- Cytoperm) before staining for intracellular cytokines (TNFa- ther dissected into three arms (either untreated, treated with APC and IFNg-FITC (both BD Biosciences). In addition, for LDBCMapa or 1 mg/mL LPS) and cultured for 48 h as depicted activation status, appropriate wells were stained with a separate in Fig. 5A. LDBCMapa treatment comprised 1 nM btz and antibody panel for NK, CD4 and CD8 T cell markers combined 1 mg/mL Mapa. Cells were harvested at 4 and 48 h to assess with activation markers (CD3-BV711, CD4-BV605 (Biole- MoDC maturation at an early vs. late timepoint. Maturation was gend), CD8-PerCPCy5.5, CD14/CD19-APCH7, CD56-PE, assessed by FACS following labeling with antibodies to CD80, CD16-FITC, CD69-PECy7, CD25-APC (BD Biosciences). CD40, CD83 and HLADR. In addition, culture supernatant Labeled cells were acquired on the BD Fortessa FACS analyzer, was harvested at 0, 4, 8 and 24 h and a multiplex Luminex assay FACS data was then analyzed using FlowJo analysis software and performed to assess changes in cytokine levels (GM-CSF, IL-4, a gating strategy (depicted in Fig. S9) to derive the percentage of IL-8, IL-12p70, IL-1b, IL-6, IL-10 and sCD40L) in response to NK and T cell subsets expressing activation markers (CD25 and the drug treatment. CD69), the degranulation marker (CD107a) and cytokines IFNg and TNF-a. Effect of drug treatment on human myeloma cell immunogenicity and NK-activating ligand expression levels Detection of NK or T cell subset proliferation following U266 and RPMI8226 myeloma cells were either untreated, or co-culture with myeloma-pulsed MoDCs treated with LDB, Mapa or LDBCMapa for 48 h (as per meth- Day 5 MoDCs were pulsed with drug-treated U266 cells at a ods above) and the effect of drug treatment on myeloma cell 1:1 ratio as in the protocol for CTL expansion culture (as immunogenicity assessed. Treated myeloma cells were labeled described above). After 4 h, cells were washed twice with culture with antibodies to CD80-FITC, CD86-PE, CD83-APC, HLA- media and left overnight. Autologous PBLs (from two separate DR-APC-H7 (all from BD Biosciences) and HLA-ABC-V450 normal donors) were washed, counted and stained with 2.5uM (e-Bioscience) and changes in protein expression detected by CTV (Life Technologies) for 20 min, washed and added to the FACS analysis. In addition, treated myeloma cells were labeled myeloma pulsed MoDCs and cultured for 7 d at a 5:1 ratio in with the antibodies CD155-FITC, MIC-A-PE, MIC-B-APC, 0.5 mL RPMI1640 plus 10% FCS, IL-2 (20 U/mL) in 48-well CD112-FITC, ULBP-2,5,6-APC, ULBP-1-PE (all RnD Sys- plates. On day 4, culture media was replaced with fresh RPMI tems) and changes in NK-activating ligand expression assessed by plus 10% FCS and low dose IL-2 (20 U/mL). Co-cultured cells FACS analysis. were harvested on day 7 and stained for NK, CD4, CD8 T cell and activation markers (CDD3-BV785, CD4-BV605 (both Assessment of direct activation of NK and T cells by drug from Biolegend), CD8-PerCP Cy5.5, CD14/CD19-APCH7, treated myeloma cells CD16-FITC, CD56-PE, CD69-PECy7, and CD25-APC (all Drug-treated U266 cells (prepared as above) were harvested at from BD Biosciences). Cells were analyzed by FACS and the level 48 h, washed twice in media and then co-cultured with HLA- of activation and proliferation assessed on NK and T cell subsets A2C PBLs. The PBLs from two normal donors were co-cultured using the same gating strategy depicted in Fig. S9. with treated U266 cells in separate cultures in triplicate wells. Cells were harvested 4 or 24 h later and labeled with antibodies Detection of expansion culture NK and T cell effector status to CD45-BV510, CD3-BV711, CD4-BV605 (Biolegend) Day 7 co-cultured cells (from above) were re-stimulated with CD14- and CD19-APCH7, CD56-PE, CD8-PECy5.5, CD25- antigen (untreated or treated U266 cells) for 4 h prior to stain- APC, CD69-PECy7 (BD Biosciences). Labeled cells were then ing. Golgi stop and CD107a-PeCy5 (BD) antibody were added

e1038011-14 OncoImmunology Volume 4 Issue 9 to the appropriate wells. Staining panels were the same as for the Supplemental Material NK, CD4 and CD8 activation protocol (above). Supplemental data for this article can be accessed on the publisher’s website. Disclosure of Potential Conflicts of Interest No potential conflicts of interest were disclosed.

Funding This work was completed with funding from Human Ethics Statement Genome Sciences Inc. and National Health and Medical Normal donor cells were acquired from Red Cross Blood Research Council of Australia (program grants 454569 and Bank donors following approval from the Peter MacCallum Can- 1013667). cer Center Human Ethics Committee.

References 11. Tesniere A, Apetoh L, Ghiringhelli F, Joza N, Panaretakis daratumumab (DARA) as monotherapy in patients 1. Attal M, Harousseau JL. Autologous peripheral blood T, Kepp O, Schlemmer F, Zitvogel L, Kroemer G. Immu- with relapsed or refractory multiple myeloma (RR progenitor cell transplantation for multiple myeloma. nogenic cancer cell death: a key-lock paradigm. Curr MM). ASCO Meet Abs 2014; 32:8513 Baillieres Best Pract Res Clin Haematol 1999; 12:171- Opin Immunol 2008; 20:504-11; PMID:18573340; 20. Plesner T, Arkenau H-T, Lokhorst HM, Gimsing P, 91; PMID:11000991; http://dx.doi.org/10.1053/beha. http://dx.doi.org/10.1016/j.coi.2008.05.007 Krejcik J, Lemech CR, Minnema M, Lassen UN, 1999.0015 12. Hsi ED, Steinle R, Balasa B, Szmania S, Draksharapu Ahmadi T, Yeh H et al. Safety and efficacy of daratu- 2. Child JA, Morgan GJ, Davies FE, Owen RG, Bell SE, A, Shum BP, Huseni M, Powers D, Nanisetti A, Zhang mumab with lenalidomide and dexamethasone in Hawkins K, Brown J, Drayson MT, Selby PJ. High- Y et al. CS1, a potential new therapeutic antibody tar- relapsed or relapsed, refractory multiple myeloma. dose chemotherapy with hematopoietic stem-cell rescue get for the treatment of multiple myeloma. Clin Cancer ASCO Meet Abstr 2014; 32:8533 for multiple myeloma. N Engl J Med 2003; 348:1875- Res 2008; 14:2775-84; PMID:18451245; http://dx. 21. Ashkenazi A, Pai RC, Fong S, Leung S, Lawrence DA, 83; PMID:12736280; http://dx.doi.org/10.1056/ doi.org/10.1158/1078-0432.CCR-07-4246 Marsters SA, Blackie C, Chang L, McMurtrey AE, NEJMoa022340 13. Tai Y-T, Dillon M, Song W, Leiba M, Li X-F, Burger Hebert A et al. Safety and antitumor activity of recom- 3. Palumbo A, Anderson K. Multiple myeloma. N Engl J P, Lee AI, Podar K, Hideshima T, Rice AG et al. Anti- binant soluble Apo2 ligand. J Clin Invest 1999; Med 2011; 364:1046-60; PMID:21410373; http://dx. CS1 humanized monoclonal antibody HuLuc63 inhib- 104:155-62; PMID:10411544; http://dx.doi.org/ doi.org/10.1056/NEJMra1011442 its myeloma cell adhesion and induces antibody-depen- 10.1172/JCI6926 4. Ocio EM, Richardson PG, Rajkumar SV, Palumbo A, dent cellular cytotoxicity in the bone marrow milieu. 22. Evdokiou A, Bouralexis S, Atkins GJ, Chai F, Hay S, Mateos MV, Orlowski R, Kumar S, Usmani S, Roodman Blood 2008; 112:1329-37; PMID:17906076; http:// Clayer M, Findlay DM. Chemotherapeutic agents sen- D, Niesvizky R et al. New drugs and novel mechanisms dx.doi.org/10.1182/blood-2007-08-107292 sitize osteogenic sarcoma cells, but not normal human of action in multiple myeloma in 2013: a report from the 14. de Weers M, Tai YT, van der Veer MS, Bakker JM, bone cells, to Apo2L/TRAIL-induced apoptosis. Int J international myeloma working group (IMWG). Leuke- Vink T, Jacobs DC, Oomen LA, Peipp M, Valerius T, Cancer 2002; 99:491-504; PMID:11992538; http:// mia 2014; 28:525-42; PMID:24253022; http://dx.doi. Slootstra JW et al. Daratumumab, a novel therapeutic dx.doi.org/10.1002/ijc.10376 org/10.1038/leu.2013.350 human CD38 monoclonal antibody, induces killing of 23. Kothny-Wilkes G, Kulms D, Poppelmann B, Luger 5. Donato F, Gay F, Bringhen S, Troia R, Palumbo A. multiple myeloma and other hematological tumors. J TA, Kubin M, Schwarz T. Interleukin-1 protects trans- Monoclonal antibodies currently in Phase II and III tri- Immunol 2011; 186:1840-8; PMID:21187443; http:// formed keratinocytes from tumor necrosis factor- als for multiple myeloma. Expert Opin Biol Ther dx.doi.org/10.4049/jimmunol.1003032 related apoptosis-inducing ligand. J Biol Chem 1998; 2014; 14:1127-44; PMID:24749510; http://dx.doi. 15. Nijhof IS, Groen RW, Noort WA, van Kessel B, de 273:29247-53; PMID:9786937; http://dx.doi.org/ org/10.1517/14712598.2014.908848 Jong-Korlaar RA, Bakker JM, Lammerts-van Bueren 10.1074/jbc.273.44.29247 6. Brimnes MK, Svane IM, Johnsen HE. Impaired func- JJ, Parren PW, Lokhorst HM, van de Donk NW et al. 24. Lawrence D, Shahrokh Z, Marsters S, Achilles K, Shih tionality and phenotypic profile of dendritic cells from Preclinical evidence for the therapeutic potential of D, Mounho B, Hillan K, Totpal K, DeForge L, Schow patients with multiple myeloma. Clin Exp Immunol CD38-targeted immuno-chemotherapy in multiple P et al. Differential hepatocyte toxicity of recombinant 2006; 144:76-84; PMID:16542368; http://dx.doi.org/ myeloma patients refractory to lenalidomide and borte- Apo2L/TRAIL versions. Nat Med 2001; 7:383-5; 10.1111/j.1365-2249.2006.03037.x zomib. Clin Cancer Res 2014; 21(12):1–9; PMID:11283636; http://dx.doi.org/10.1038/86397 7. Schutt P, Brandhorst D, Stellberg W, Poser M, Ebeling PMID:25398450 25. Menoret E, Gomez-Bougie P, Geffroy-Luseau A, Dan- P, Muller S, Buttkereit U, Opalka B, Lindemann M, 16. van der Veer MS, de Weers M, van Kessel B, Bakker iels S, Moreau P, Le Gouill S, Harousseau JL, Bataille Grosse-Wilde H et al. Immune parameters in multiple JM, Wittebol S, Parren PW, Lokhorst HM, Mutis T. R, Amiot M, Pellat-Deceunynck C. Mcl-1L cleavage is myeloma patients: influence of treatment and correla- Towards effective immunotherapy of myeloma: involved in TRAIL-R1- and TRAIL-R2-mediated apo- tion with opportunistic infections. Leuk Lymphoma enhanced elimination of myeloma cells by combination ptosis induced by HGS-ETR1 and HGS-ETR2 human 2006; 47:1570-82; PMID:16966269; http://dx.doi. of lenalidomide with the human CD38 monoclonal mAbs in myeloma cells. Blood 2006; 108:1346-52; org/10.1080/10428190500472503 antibody daratumumab. Haematologica 2011; 96:284- PMID:16638930; http://dx.doi.org/10.1182/blood- 8. Chan AC, Neeson P, Leeansyah E, Tainton K, Quach 90; PMID:21109694; http://dx.doi.org/10.3324/ 2005-12-007971 H, Prince HM, Harrison SJ, Godfrey DI, Ritchie D, haematol.2010.030759 26. Mitsiades CS, Treon SP, Mitsiades N, Shima Y, Berzins SP. Natural killer T cell defects in multiple 17. Jakubowiak AJ, Benson DM, Bensinger W, Siegel DS, Richardson P, Schlossman R, Hideshima T, Anderson myeloma and the impact of lenalidomide therapy. Clin Zimmerman TM, Mohrbacher A, Richardson PG, KC. TRAIL/Apo2L ligand selectively induces apoptosis Exp Immunol 2014; 175:49-58; PMID:24032527; Afar DE, Singhal AK, Anderson KC. Phase I trial of and overcomes drug resistance in multiple myeloma: http://dx.doi.org/10.1111/cei.12196 anti-CS1 monoclonal antibody elotuzumab in combi- therapeutic applications. Blood 2001; 98:795-804; 9. Hsu AK, Quach H, Tai T, Prince HM, Harrison SJ, nation with bortezomib in the treatment of relapsed/ PMID:11468181; http://dx.doi.org/10.1182/blood. Trapani JA, Smyth MJ, Neeson P, Ritchie DS. The refractory multiple myeloma. J Clin Oncol 2012; V98.3.795 immunostimulatory effect of lenalidomide on NK-cell 30:1960-5; PMID:22291084; http://dx.doi.org/ 27. Belch A, Sharma A, Spencer A, Tarantolo S, Bahlis NJ, function is profoundly inhibited by concurrent dexa- 10.1200/JCO.2011.37.7069 Doval D, Gallant G, Kumm E, Klein J, Chanan-Khan methasone therapy. Blood 2011; 117:1605-13; 18. Lonial S, Vij R, Harousseau JL, Facon T, Moreau P, AA. A multicenter randomized phase II trial of mapatu- PMID:20978269; http://dx.doi.org/10.1182/blood- Mazumder A, Kaufman JL, Leleu X, Tsao LC, West- mumab, a TRAIL-R1 agonist monoclonal antibody, in 2010-04-278432 land C et al. Elotuzumab in combination with lenali- combination with bortezomib in patients with 10. Rapoport AP, Stadtmauer EA, Aqui N, Badros A, Cotte J, domide and low-dose dexamethasone in relapsed or relapsed/refractory multiple myeloma (MM). ASH Chrisley L, Veloso E, Zheng Z, Westphal S, Mair R et al. refractory multiple myeloma. J Clin Oncol 2012; Ann Meet Abstr 2010; 116:5031 Restoration of immunity in lymphopenic individuals 30:1953-9; PMID:22547589; http://dx.doi.org/ 28. Straube C, Wehner R, Wendisch M, Bornhauser M, with cancer by vaccination and adoptive T-cell transfer. 10.1200/JCO.2011.37.2649 Bachmann M, Rieber EP, Schmitz M. Bortezomib sig- Nat Med 2005; 11:1230-7; PMID:16227990; http://dx. 19. Lokhorst HM, Laubach J, Nahi H, Plesner T, Gimsing nificantly impairs the immunostimulatory capacity of doi.org/10.1038/nm1310 P, Hansson M, Minnema M, Lassen UN, Krejcik J, human myeloid blood dendritic cells. Leukemia 2007; Ahmadi T et al. Dose-dependent efficacy of

www.tandfonline.com OncoImmunology e1038011-15 21:1464-71; PMID:17495970; http://dx.doi.org/ multiple myeloma: the role of interleukin-6. Blood 2002; 195:1653-9; PMID:12070293; http://dx.doi. 10.1038/sj.leu.2404734 2002; 100:230-7; PMID:12070032; http://dx.doi.org/ org/10.1084/jem.20020338 29. Subklewe M, Sebelin-Wulf K, Beier C, Lietz A, Mathas 10.1182/blood.V100.1.230 42. Dhodapkar KM, Kaufman JL, Ehlers M, Banerjee DK, S, Dorken B, Pezzutto A. Dendritic cell maturation 35. Yang D-H, Park J-S, Jin C-J, Kang H-K, Nam J-H, Bonvini E, Koenig S, Steinman RM, Ravetch JV, Dho- stage determines susceptibility to the proteasome inhib- Rhee J-H, Kim Y-K, Chung S-Y, Choi S-J-N, Kim H-J dapkar MV. Selective blockade of inhibitory Fcgamma itor bortezomib. Hum Immunol 2007; 68:147-55; et al. The dysfunction and abnormal signaling pathway receptor enables human dendritic cell maturation with PMID:17349869; http://dx.doi.org/10.1016/j. of dendritic cells loaded by tumor antigen can be over- IL-12p70 production and immunity to antibody- humimm.2006.12.005 come by neutralizing VEGF in multiple myeloma. Leu- coated tumor cells. Proc Natl Acad Sci U S A 2005; 30. Blanco B, Perez-Simon JA, Sanchez-Abarca LI, Carva- kemia Res 2009; 33:665-70; PMID:18922577; http:// 102:2910-5; PMID:15703291; http://dx.doi.org/ jal-Vergara X, Mateos J, Vidriales B, Lopez-Holgado dx.doi.org/10.1016/j.leukres.2008.09.006 10.1073/pnas.0500014102 N, Maiso P, Alberca M, Villaron E et al. Bortezomib 36. Barreira da Silva R, Munz€ C. Natural killer cell activa- 43. Dauer M, Obermaier B, Herten J, Haerle C, Pohl K, induces selective depletion of alloreactive T lympho- tion by dendritic cells: balancing inhibitory and activat- Rothenfusser S, Schnurr M, Endres S, Eigler A. Mature cytes and decreases the production of Th1 cytokines. ing signals. Cell Mol Life Sci 2011; 68:3505-18; dendritic cells derived from human monocytes within Blood 2006; 107:3575-83; PMID:16282346; http:// PMID:21861182; http://dx.doi.org/10.1007/s00018- 48 hours: a novel strategy for dendritic cell differentia- dx.doi.org/10.1182/blood-2005-05-2118 011-0801-8 tion from blood precursors. J Immunol 2003; 31. Chanan-Khan A, Sonneveld P, Schuster MW, Stadt- 37. Fauriat C, Long EO, Ljunggren HG, Bryceson YT. 170:4069-76; PMID:12682236; http://dx.doi.org/ mauer EA, Facon T, Harousseau J-L, Ben-Yehuda D, Regulation of human NK-cell cytokine and chemokine 10.4049/jimmunol.170.8.4069 Lonial S, Goldschmidt H, Reece D et al. Analysis of production by target cell recognition. Blood 2010; 44. Sallusto F, Lanzavecchia A. Efficient presentation of soluble herpes zoster events among bortezomib-treated patients 115:2167-76; PMID:19965656; http://dx.doi.org/ antigen by cultured human dendritic cells is maintained by in the phase III APEX study. J Clin Oncol 2008; 10.1182/blood-2009-08-238469 granulocyte/macrophage colony-stimulating factor plus 26:4784-90; PMID:18711175; http://dx.doi.org/ 38. Piccioli D, Sbrana S, Melandri E, Valiante NM. Con- interleukin 4 and downregulated by tumor necrosis factor 10.1200/JCO.2007.14.9641 tact-dependent stimulation and inhibition of dendritic alpha. J Exp Med 1994; 179:1109-18; PMID:8145033; 32. LeBlanc R, Catley LP, Hideshima T, Lentzsch S, Mit- cells by natural killer cells. J Exp Med 2002; 195:335- http://dx.doi.org/10.1084/jem.179.4.1109 siades CS, Mitsiades N, Neuberg D, Goloubeva O, 41; PMID:11828008; http://dx.doi.org/10.1084/ 45. Day D, Pham K, Ludford-Menting MJ, Oliaro J, Izon Pien CS, Adams J et al. Proteasome inhibitor PS-341 jem.20010934 D, Russell SM, Gu M. A method for prolonged imag- inhibits human myeloma cell growth in vivo and pro- 39. Walzer T, Dalod M, Robbins SH, Zitvogel L, Vivier E. ing of motile lymphocytes. Immunol Cell Biol 2009; longs survival in a murine model. Cancer Res 2002; Natural-killer cells and dendritic cells: “l’union fait la 87:154-8; PMID:18982018; http://dx.doi.org/ 62:4996-5000; PMID:12208752 force”. Blood 2005; 106:2252-8; PMID:15933055; 10.1038/icb.2008.79 33. Surget S, Chiron D, Gomez-Bougie P, Descamps G, http://dx.doi.org/10.1182/blood-2005-03-1154 46. Adams J, Palombella VJ, Sausville EA, Johnson J, Des- Menoret E, Bataille R, Moreau P, Le Gouill S, Amiot 40. Walzer T, Dalod M, Vivier E, Zitvogel L. Natural killer tree A, Lazarus DD, Maas J, Pien CS, Prakash S, Elliott M, Pellat-Deceunynck C. Cell death via DR5, but not cell-dendritic cell crosstalk in the initiation of immune PJ. Proteasome inhibitors: a novel class of potent and DR4, is regulated by p53 in myeloma cells. Cancer Res responses. Exp Opin Biol Ther 2005; 5(Suppl 1):S49- effective antitumor agents. Cancer Res 1999; 59:2615- 2012; 72:4562-73; PMID:22738917; http://dx.doi. 59; PMID:16187940; http://dx.doi.org/10.1517/ 22; PMID:10363983 org/10.1158/0008-5472.CAN-12-0487 14712598.5.1.S49 47. Lightcap ES, McCormack TA, Pien CS, Chau V, 34. Ratta M, Fagnoni F, Curti A, Vescovini R, Sansoni P, 41. Kalergis AM, Ravetch JV. Inducing tumor immunity Adams J, Elliott PJ. Proteasome inhibition measure- Oliviero B, Fogli M, Ferri E, Della Cuna GR, Tura S through the selective engagement of activating ments: clinical application. Clin Chem 2000; 46:673- et al. Dendritic cells are functionally defective in Fcgamma receptors on dendritic cells. J Exp Med 83; PMID:10794750

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