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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Mol Cancer Ther. Author manuscript; available in PMC Sep 1, 2010.
Published in final edited form as:
PMCID: PMC2748787
NIHMSID: NIHMS140372
Combinatorial efficacy of anti-CS1 monoclonal antibody elotuzumab (HuLuc63) and bortezomib against multiple myeloma
Frits van Rhee,1 Susann M. Szmania,1 Myles Dillon,2 Anne M. van Abbema,2 Xin Li,1 Mary K. Stone,1 Tarun K Garg,1 JuMei Shi,1 Amberly M. Moreno-Bost,1 Rui Yun,2 Balaji Balasa,2 Bishwa Ganguly,2 Debra Chao,2 Audi G. Rice,2 Fenghuang Zhan,1 John D. Shaughnessy, Jr,1 Bart Barlogie,1 Shmuel Yaccoby,1 and Daniel E.H. Afar2
1Myeloma Institute for Research and Therapy, University of Arkansas for Medical Sciences, Little Rock, AR
2Department of Research, Facet Biotech Corp., Redwood City, CA
Corresponding author: Frits van Rhee, University of Arkansas for Medical Sciences, Myeloma Institute for Research and Therapy, 4301 West Markham, # 776, Little Rock AR 72205. Phone: 501-526-6990, ext. 2425, Fax: 501-526-5075, vanrheefrits/at/uams.edu
Monoclonal antibody (mAb) therapy for multiple myeloma, a malignancy of plasma cells, has not been clinically efficacious in part due to a lack of appropriate targets. We recently reported that the cell surface glycoprotein CS1 (CD2 subset 1, CRACC, SLAMF7, CD319), was highly and universally expressed on myeloma cells while having restricted expression in normal tissues. Elotuzumab (formerly known as HuLuc63), a humanized mAb targeting CS1, is currently in a Phase I clinical trial in relapsed/refractory myeloma. In this report we investigated whether the activity of elotuzumab could be enhanced by bortezomib, a reversible proteasome inhibitor with significant activity in myeloma. We first showed that elotuzumab could induce patient-derived myeloma cell killing within the bone marrow microenvironment using a SCID-hu mouse model. We next showed that CS1 gene and cell surface protein expression persisted on myeloma patient-derived plasma cells collected after bortezomib administration. In vitro bortezomib pretreatment of myeloma targets significantly enhanced elotuzumab-mediated antibody-dependent cell-mediated cytotoxicity (ADCC), both for OPM2 myeloma cells using natural killer (NK) or peripheral blood mononuclear cells (PBMC) from healthy donors and for primary myeloma cells using autologous NK effector cells. In an OPM2 myeloma xenograft model, elotuzumab in combination with bortezomib exhibited significantly enhanced in vivo anti-tumor activity. These findings provide the rationale for a clinical trial combining elotuzumab and bortezomib, which will test the hypothesis that combining both drugs would result in enhanced immune lysis of myeloma by elotuzumab and direct targeting of myeloma by bortezomib.
Keywords: multiple myeloma, CS1, bortezomib, antibody therapy, natural killer cells
Considerable progress has been made in the management of multiple myeloma (MM) by the application of high dose chemotherapy supported by autologous stem cell transplantation, which prolongs overall and disease free survival.1 We have recently reported that combining novel agents such as thalidomide and bortezomib with stem cell transplantation can substantially improve outcome and overcome the poor prognosis imparted by the FGFR3/ MMSET translocation.and17p deletion.2 Monoclonal antibody (mAb) therapy is non-cross resistant with chemotherapy and has the potential for further improving results as has been the case for rituximab in B-cell non-Hodgkin lymphomas. The CD-2 subgroup of myeloma frequently over expresses CD20,3 but therapy with rituximab has yielded disappointing results probably due to heterogeneous expression of CD20 and the presence of the complement regulator CD59 on myeloma which renders complement mediated cytotoxicity ineffective.4,5,6,7
At present there are no mAbs approved for the treatment of MM. A number of mAbs are under investigation in MM targeting antigens such as CD40,8-10 CD56,11 CD74,12,13 and HM1.24.14 We have recently reported that the cell surface glycoprotein CS1 (CD2 subset 1, CRACC, SLAMF7, CD319), a member of the signaling lymphocyte activating-molecule-related receptor family, is selectively and uniformly expressed at high levels on myeloma cells independent of the presence of metaphase cytogenetic abnormalities or molecular subgroup.15 CS1 is not expressed by normal tissues or stem cells, with the exception of some lymphocyte subsets which have lower expression levels compared to malignant plasma cells.15 Interestingly, the CS1 gene is located on chromosome 1q, amplifications of which are frequent in aggressive myeloma and linked to early myeloma related death due to over expression of the cell cycle regulator CKS1B.16
We and others have demonstrated that the humanized anti-CS1 mAb elotuzumab exerts anti-myeloma activity in vitro via antibody-dependent cellular cytotoxicity (ADCC) mediated by NK cells and does not rely on complement mediated cytotoxicity.15,17 Elotuzumab has single agent activity in myeloma xenograft models and is currently being tested in a phase I safety trial for relapsed/refractory myeloma.15,17,18 Recent work shows that down-regulation of the cell surface expression of MHC Class I, an inhibitor of NK cell function, by bortezomib enhances the susceptibility of myeloma cells to NK cell-mediated killing.19 Furthermore, bortezomib-induced up regulation of CD95 and TRAILR2 on tumor cells can lead to enhanced NK cell mediated killing.20 We therefore hypothesized that the combination of elotuzumab and bortezomib may maximize NK cell mediated destruction of MM. In the present study, we first establish that elotuzumab is effective in the SCID-hu mouse model which allows for the growth of primary myeloma cells in a human bone micro-environment. Further, we demonstrate that combining elotuzumab with bortezomib leads to strong anti-myeloma activity both in vitro and in vivo.
Cells, cell lines, and cultures
Peripheral blood was collected from healthy donors, and fresh blood or bone marrow was collected from patients with MM after informed consent in accordance with the Declaration of Helsinki. Approval was obtained from the University of Arkansas for Medical Sciences Institutional Review Board for these studies. NK cells were enriched from whole blood samples either with the RosetteSep human NK cell enrichment cocktail (StemCell Technologies Inc., Vancouver, BC) or using microbeads coated with CD56 mAb (Miltenyi Biotech, Auburn, CA). CD138-positive plasma cells were purified from patient-derived bone marrow aspirates via positive selection with anti-CD138 microbeads (Miltenyi Biotech). After magnetic bead selection, cells were confirmed to be >90% pure by flow cytometry for CD56 or CD138, respectively. The OPM2 and K562 cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA).
Reagents
Clinical grade bortezomib (VELCADE®) was purchased from or gifted by Millennium Pharmaceuticals, Inc., (Cambridge, MA). Anti-human CS1 mAbs were generated as previously described, including murine IgG2a MuLuc63 and its humanized IgG1 version elotuzumab (formerly HuLuc63) both specific for the extracellular region of human CS1, and 1G9 mAb which recognizes the intracellular region.15 The human IgG1 isotype control mAb, MSL109, used in all in vitro experiments, is a fully human anti-cytomegalovirus mAb.21 Murine IgG2a isotype control mAb used for all in vivo experiments is a mAb directed against VP7 of bluetongue virus (BTV) and is available as a hybridoma from the ATCC (clone 8A3B.6).
SCID-hu mouse model
C.B-17/IcrHsd-Prkdcscid (Harlan Sprague, Indianapolis, Indiana) were implanted with human fetal bone (Advanced Bioscience Resources, Alameda, CA) subcutaneously as previously described.22,23 Upon bone engraftment, 2-8 × 106 cells from heparinized bone marrow aspirates obtained from myeloma patients containing at least 20% CD138+ plasma cells were injected directly into the human fetal bone in 50 μL phosphate-buffered saline (PBS). For each experiment, cells from the same patient were injected into the fetal bone of 5 to 6 SCID-hu hosts. Changes in levels of circulating human immunoglobulin (hu Ig) of the M-protein isotype, monitored by ELISA as previously described,22 was used as an indicator of myeloma growth. When hu Ig levels reached 10 μg/mL or higher in 2 consecutive measurements, 2 mice with comparable tumor load (tumor derived from the same patient) were randomized to receive murine isotype control mAb (anti-BTV) or MuLuc63 (parental mouse anti-CS1 mAb). Dosing was 10 mg/kg of mAb administered intraperitoneally two times a week for a total of 6 doses. Tumor growth was monitored for a period of about 6 weeks by ELISA for hu Ig. The statistical significance of differences was assessed with the student t test; P < 0.05 was considered significant. All animal work was carried out according to the National Institutes of Health's Guide for the Care and Use of Laboratory Animals, with protocols approved by the University of Arkansas for Medical Sciences Institutional Animal Care and Use Committee.
Gene expression profiling
CD138-positive plasma cells were purified from samples of bone marrow from patients with MM, as described above, before and 48 hours (h) after a single dose of 1.0mg/m2 bortezomib.2 Gene expression profiling for CS1 expression (probe set 219159_s_at) was performed using the Affymetrix U133 Plus 2.0 arrays platform (Affymetrix Inc. Santa Clara, CA) as previously described.24 The student's t test was used to determine whether CS1 gene expression was significantly different in plasma cells collected before versus after bortezomib administration.
Flow cytometry
CD138-purified MM cells from bone marrow collected from patients before and 48h after a dose of 1.0mg/m2 bortezomib were purified as above, cryopreserved, then subsequently thawed and analyzed in the same experiment by flow cytometry for CS1 expression. Dead and apoptotic cells were gated out using propidium iodide and annexin V stains (Invitrogen, Carlsbad, CA) and the resulting viable cell population was analyzed for CS1 expression using elotuzumab conjugated to fluorescein isothiocyanate (FITC). Samples were run on a FACSARIA cytometer (BD Pharmingen, San Diego, CA). Histogram overlays were created using FCS express software (De Novo Software, Los Angeles, CA).
Antibody-dependent cellular cytotoxicity (ADCC) assay
Target cells were suspended at a density of 1.0 × 106 cells/mL in RPMI media with a fixed dose of 10 nM bortezomib or titrated doses of bortezomib (20 nM, 10 nM, 5 nM, 1 nM) or in media alone and incubated in a 5% CO2 incubator at 37° for 18 h. Cells were collected, washed, assessed for viability, re-suspended at a density of 20 × 106 viable cells/mL, and labeled for 1 h with 50 mCi Na2 [51Cr]O4 per 106 cells. 51Cr-labeled cells were washed and added to a 96-well, V-bottomed polystyrene plate at a cell density of 15,000 cells per 75 μL. Elotuzumab or MSL109 (isotype control mAb) was added in a volume of 25 μl to the target cells for a final concentration of 0.001 to 10 μg/mL. NK cells or PBMC from healthy donors were added in a volume of 100 μl to bortezomib-treated or mock-treated target cells at a ratio of 10:1 (NK:targets) or 25:1 (PBMC:targets). Pretreatment of targets for 0.5 h with 50 mcg/ml Fc receptor blocking antibody (Serotec, Oxford, UK) was performed for some assays. After a 4-hour incubation at 37°C, cells were centrifuged at 1200 rpm for 5 minutes, and released 51Cr was measured in the 100 uL supernatants. Maximum release was determined from target cells lysed with 100 mg/ml digitonin or 1% Triton X-100. Antibody independent cellular cytotoxicity (AICC) was determined using target cells, plus media, plus NK cells; spontaneous lysis was determined using 51Cr-labeled cells plus media without NK or PBMC effectors. Percent cytotoxicity was calculated as ([sample - AICC]/[Maximum - AICC]) * 100. The concentration of elotuzumab necessary to produce 50% of the maximal elotuzumab-mediated ADCC response (EC50) was determined from a curve fit using the sigmoidal dose-response non-linear curve regression in GraphPad Prism. EC50 values from the untreated cells were compared to those cells treated with bortezomib in the same experiment. A two tailed paired t-test was used to determine the p value.
Primary myeloma cells were purified from patient bone marrow, as described above, and treated with 5 or 20 nM bortezomib or vehicle for 18 hours to assess the effects of bortezomib on elotuzumab-mediated lysis of patient-derived autologous NK cells. Cr51 release assays were performed as above with NK cell : myeloma cell ratios of 1:1, 10:1, and 30:1. Elotuzumab and isotype control mAb MSL109 were used at a concentration of 10 μg/mL. The NK cell-sensitive cell line K562 was included as a positive control. Percent cytotoxicity was calculated as ([sample - spontaneous lysis]/[Maximum - spontaneous lysis]) * 100. In vitro experiments were performed in triplicate, and the results are reported as mean ± standard error of the mean (SEM).
Mouse xenograft model
Female IcrTac:ICR-Prkdcscid mice (6-8 weeks of age) were obtained from Taconic Farms (Germantown, NY) and were inoculated in the lower right flank with 1 × 107 OPM2 cells (American Type Culture Collection, Manassas, VA) in RPMI-1640 media from HyClone (Logan, UT). Caliper measurements were performed thrice weekly for the calculation of tumor volume, and tumor growth was monitored for a period of 1 to 2 months. The following formula was used to calculate tumor volume: L×W×H/2, where L (length) is the longest side of the tumor in the plane of the animal's back; W (width) is the longest measurement perpendicular to the length and in the same plane; and H (height) is the highest point perpendicular to the back of the animal.
Mice with an average tumor size of 100 mm3 were allocated randomly into treatment groups of 10-15 mice each. Bortezomib was administered intraperitoneally in phosphate-buffered saline (PBS) at a dose of 1 mg/kg or twice weekly for weeks 1 and 2, no treatment for week 3, and 1 mg/kg twice weekly for weeks 4 and 5 (total of 8 doses). Elotuzumab or MSL 109 (at doses of 1 or 10 mg/kg in PBS) were administered intraperitoneally twice weekly for 5 weeks (total of 10 doses) on days offset from bortezomib dosing. One-way analysis of variance with a Tukey post-test was used to determine differences between elotuzumab and control treatment groups. All animal work was carried out according to the National Institutes of Health's Guide for the Care and Use of Laboratory Animals, with protocols approved by the Institutional Animal Care and Use Committee at PDL BioPharma.
Immunohistochemistry (IHC)
Engrafted tumor with human fetal bones in the SCID-Hu model were removed, formalin fixed for 24 h and then decalcified with EDTA, pH 7.0. Decalcified, formalin-fixed, paraffin-embedded tissues were cut into 4μM sections for Hematoxylin and Eosin (H&E) staining. OPM2 xenograft tumors were harvested at the end of study and fixed in formalin for 24 hours. De-paraffined tissue slides were immersed in DAKO Target retrieval solution (pH 6) in a decloaking chamber (Biocare Medical, Concord CA) for antigen epitope retrieval. CS1 expression in OPM2 xenografts and primary myeloma tumor grafts was determined by staining the tissues with 1G9 mouse mAb at 1 μg/ml for 30minutes and EnVision® secondary polymer System (Dako, Carpinteria, CA). Magnification is at 50×. Percent tumor burden was determined by a certified pathologist using histological analysis of the tumor grafts.
Elotuzumab induces killing of primary myeloma cells in the SCID-hu mouse model
Elotuzumab exhibits significant in vivo anti-tumor activity towards myeloma cell lines, subcutaneously inoculated in SCID mice.17,15 To determine whether anti-CS1 therapy can inhibit the growth of primary myeloma cells in the human bone microenvironment, primary myeloma cells were injected into subcutaneously implanted human fetal bones engrafted in SCID mice. For each experiment 5 to 6 SCID-hu mice were injected with bone marrow from the same patient allowing for the selection of two mice with comparable tumor volumes, which were randomly allocated to MuLuc63 (the parental mAb from which elotuzumab was derived) or control mAb treatment. The mouse parental MuLuc63 mAb was used in these experiments to facilitate the measurement of circulating human IgG in the mice. We observed a statistically significant reduction of tumor volume and circulating human IgG levels in mice treated with MuLuc63, compared to the isotype control mAb (Fig. 1). Two of the 4 patients studied (subjects 2 and 3) had high risk myeloma based on the 70 gene model and responded equally well to MuLuc63 monotherapy as low risk disease (subjects 1 and 4).16 Histological analysis of the fetal bones (in 3/3 evaluable cases) demonstrated nearly complete eradication of MM in the MuLuc63 group, whilst MM effaced the marrow in the control mAb group (Fig. 2). These results with MuLuc63 infer that elotuzumab would exhibit significant anti-myeloma activity in the bone marrow microenvironment.
Figure 1
Figure 1
Anti-CS1 mAb MuLuc63 induces marked reduction of primary human myeloma tumors in the SCID hu host
Figure 2
Figure 2
Histological analysis of tumor burden with anti-CS1 therapy
CS1 RNA and protein expression persists after bortezomib administration
Since one of the goals of the present study was to determine the effect of combining elotuzumab with bortezomib on anti-tumor activity, the effect of bortezomib on CS1 expression was examined. The gene expression profile of primary myeloma cells from 103 previously untreated patients both before and after a single dose of bortezomib 1mg/m2 was examined. No significant difference in mean CS1 gene expression level (p=0.96, Fig. 3A) was observed. Furthermore, flow cytometry analysis showed that bortezomib did not alter the level of CS1 protein expression at the myeloma cell surface (Fig. 3B).
Figure 3
Figure 3
Figure 3
CS1 gene expression persists in patients with MM after bortezomib treatment
Bortezomib enhances elotuzumab mediated ADCC of CS1 positive myeloma cell lines and primary myeloma cells
There was a dose dependent increase in elotuzumab-mediated ADCC of the myeloma cell line OPM2, which was enhanced by pre-treatment with 10nM bortezomib (Fig. 4A). The EC50 of elotuzumab-mediated ADCC was decreased significantly by bortezomib in 4 independent experiments with NK cells from normal donors. Bortezomib also significantly (p<0.0006) increased elotuzumab induced lysis of the CS1 positive cell line XG-1, while the CS1 negative and bortezomib resistant cell line RPMI8226/R5 was insensitive to treatment with either drug (Fig. 4B). Preincubation of effectors with FcR blocking antibody abrogated elotuzumab induced killing, supporting the ADCC mechanism. When OPM2 cells were pre-treated with varying doses of bortezomib (1, 5, 10, 20 nM) significant enhancement of elotuzumab-mediated ADCC was observed only at doses of 5 nM and above (results shown for 5 nM dose in Fig. 4C). A dose of 1 nM bortezomib had no significant effect on ADCC enhancement. Also, no significant difference was observed between 5, 10, and 20 nM suggesting that a threshold level of 5 nM bortezomib is enough for maximal enhancement for OPM2 cells (data not shown). Patient derived NK cells induced approximately 20% specific lysis of autologous myeloma cells in the presence of elotuzumab (10 μg/ml) alone at a ratio of 30 NK cells to 1 MM cell (Fig. 4D). A dose dependent increase in specific lysis was observed when adding bortezomib. The CS1-mediated lysis of primary myeloma cells was similar to killing of the NK cell sensitive cell line K562 at a dose of 20nM of bortezomib. This increase was not noted when isotype control mAb was added suggesting that bortezomib sensitized primary myeloma cells specifically to elotuzumab-mediated ADCC.
Figure 4
Figure 4
Figure 4
Figure 4
Figure 4
Bortezomib pretreatment enhances elotuzumab-mediated ADCC
Elotuzumab in combination with bortezomib exhibits significantly enhanced anti-myeloma activity towards the OPM2 xenograft model
Elotuzumab has previously been shown to exert significant dose-dependent anti-myeloma activity in mouse xenograft models.17,15 We next investigated whether elotuzumab combined with bortezomib exhibit enhanced in vivo anti-tumor activity using the OPM2 xenograft tumor model. The mice were randomized into 4 groups of 15 animals when the OPM2 tumors reached an average of 100mm3. Mice were injected intraperitoneally twice weekly with a previously established suboptimal dose of elotuzumab (1mg/kg) or control mAb. This dose is 10-fold lower than the dose of elotuzumab that achieves optimal anti-tumor activity using the OPM2 xenograft model.17 Bortezomib or PBS was administered twice weekly for weeks 1 and 2, no treatment on week 3 and twice weekly during weeks 4 and 5. The dose of bortezomib was 1 mg/kg, which when given twice a week nears the maximum tolerated dose in these mice (data not shown). There was rapid tumor growth in mice treated with control mAb. Modest, but significant anti-OPM2 activity was observed in the 2 groups treated with bortezomib alone (p<0.001) or elotuzumab alone (p<0.001). In contrast, there was a profound retardation of OPM2 tumor growth when mice were treated with elotuzumab combined with bortezomib. At day 38 of the study (the day at which the control group was terminated), mice in the combination treatment group exhibited mean tumor volumes 85% smaller than in the elotuzumab monotherapy group (p<0.001), and 82% smaller than in the bortezomib monotherapy group (p<0.001). At day 45 (the day at which the monotherapy groups were terminated), the combination therapy inhibited mean tumor volumes by 89% (P<0.001) and 87% (P<0.001) compared to elotuzumab and bortezomib monotherapy respectively. This experiment was performed three times, with similar results observed in all cases. When elotuzumab was used at the optimal dose of 10 mg/kg given twice weekly, the combination with bortezomib was not significantly more efficacious than the combination with 1 mg/kg of antibody (see supplemental data). This suggests that maximal combinatorial effect is already achieved when elotuzumab is used at the sub-optimal dose of 1 mg/kg. IHC analysis of OPM2 tumors from mice with the non-competing anti-CS1 mAb 1G9 demonstrated that the expression of CS1 protein in vivo was not affected by bortezomib treatment (data not shown), in agreement with the results shown for myeloma cells from patients treated with bortezomib. These findings substantiate the notion that elotuzumab in combination with bortezomib exhibits significantly enhanced anti-tumor activity.
CS1, a cell surface glycoprotein of the CD2 family, is highly and uniformly expressed by myeloma cells and therefore merits investigation as a potential new target for myeloma therapy. Recent studies suggest that CS1 localizes to the uropods of polarized myeloma cells suggesting a potential role for CS1 in mediating adhesion of myeloma cells to bone marrow stroma.25 CS1 also appears to protect myeloma cell lines from apoptosis by decreasing phosphorylation of ERK1/2, AKT and STAT as well as modulating other pro- and anti-apoptotic pathways.17 Preliminary data suggest that CS1 specific cytotoxic T-cells can kill both CS1 peptide pulsed T2 cells and the myeloma cell line MCCAR.26 Several lines of evidence suggest that the CS1-specific humanized mAb elotuzumab kills MM via ADCC mediated by NK cells. Blocking of the Fc receptor on NK cells abrogated the anti-myeloma effect of elotuzumab. Further, in vivo NK cell depletion in mouse xenograft models substantially reduces elotuzumab activity. Lastly, elotuzumab has no significant anti-tumor activity in NOD-SCID/IL2Rγ knock-out mice, which are deficient in NK cells.15 Since bortezomib has been shown to increase NK cell mediated lysis of primary myeloma cells by down regulating HLA-C, the principal ligand for inhibitory KIR receptors on NK cells,19 it was hypothesized that bortezomib might enhance the anti-myeloma activity of elotuzumab.
Elotuzumab delays or abrogates the growth of human myeloma cell lines in several mouse xenograft models. However, most primary myeloma cells are dependent on stimuli provided by the bone marrow micro-environment and do not grow outside the skeleton in patients. As a result it has proven very difficult to grow primary myeloma cells in SCID-hosts, which limits extrapolation of results obtained with cell lines to the human setting. We therefore first established that elotuzumab has in vivo activity against primary myeloma cells by injecting bone marrow from myeloma patients into human fetal bones engrafted into SCID-hosts. In this model, growth of the primary myeloma cells is confined to human bone and mice develop classical myeloma symptoms such as bone marrow plasmacytosis, circulating monoclonal immunglobulins and severe resorption of fetal bone.22 Administration of the parental mouse antibody of elotuzumab (MuLuc63) for only 3 weeks reduced paraprotein levels and significantly reduced tumor burden in all animals at a dose level which is clinically achievable in humans and comparable to concentrations found in the serum of patients treated with therapeutic doses of rituximab.27 Histological analysis confirmed that virtually all myeloma cells were abolished in the MuLuc63-treated animals, whilst the marrow was replaced by myeloma cells in the control mAb group.
We next examined whether the combination of elotuzumab and bortezomib has enhanced anti-myeloma activity. Administration of a single ‘test’ dose of bortezomib at 1.0mg/m2 has been shown to significantly alter the gene expression profile of both primary myeloma cells and the human bone marrow environment.28 Our results demonstrated that there was no significant alteration in CS1 RNA expression and CS1 protein expression at the cell surface after administration of clinically relevant bortezomib doses to a large cohort of untreated myeloma patients. Combination therapy of elotuzumab with bortezomib in vitro significantly lowered the EC50 of elotuzumab in experiments with the cell line OPM2 and allogeneic effector cells from normal donors. Experiments targeting the CS1 positive, bortezomib sensitive myeloma cell line XG-1 demonstrated that bortezomib significantly increased killing of elotuzumab treated targets, whereas neither drug induced lysis of the bortezomib resistant, CS1 negative myeloma line RPMI8226/R5. Further, increasing doses of bortezomib produced modest increases in killing of primary myeloma cells by autologous NK cells in the presence of control mAb. In contrast, the combination of bortezomib and elotuzumab augmented killing of primary myeloma cells to a level similar to lysis of the NK cell sensitive positive control K562. In the OPM2 xenograft model combination therapy with elotuzumab and bortezomib induced significantly better tumor growth inhibition in comparison to application of either agent alone. Taken together these data support the notion that co-treatment of elotuzumab with bortezomib produces maximal anti-myeloma activity. These findings are in keeping with recent observations by Tai et al where bortezomib enhanced elotuzumab-induced ADCC of the myeloma cell line MM1R by NK cells from healthy donors.17
In summary, this study demonstrated significant anti-CS1 mAb efficacy against patient derived myeloma tumors in a human bone microenvironment. Furthermore, bortezomib significantly potentiated the effects of elotuzumab, possibly by rendering myeloma more vulnerable to NK cell mediated lysis. These studies form the basis for clinical studies of combined elotuzumab and bortezomib therapy utilizing a novel approach in which myeloma cells are targeted directly by bortezomib, whilst at the same time enhancing the immune activity of elotuzumab. With the increased availability of drugs that target independent pathways involved in the pathophysiology of MM, the goal of future investigations will be to determine those agents with the best synergism to be used for combination therapy to provide the greatest clinical benefits for patients suffering from this disease.29,30
Figure 5
Figure 5
Bortezomib enhances elotuzumab-mediated antitumor activity in the OPM2 mouse xenograft model
Supplementary Material
Acknowledgments
The authors would also like to thank Tina Emek for excellent technical assistance, Gary Starling, Matt Williams and Debbie Law for critical review of the manuscript, and Lisa Shannon, PharmD of Scientific Connexions for editing services. This work was supported by National Institutes of Health grants CA55819 and CA134522.
FvR is supported by National Institutes of Health grants CA55819, CA134522, Multiple Myeloma Senior award 28-06 and funding from Facet Biotech Corp.
Abbreviations
mAbmonoclonal antibody
ADCCantibody dependant cellular cytotoxicity
MMmultiple myeloma
CS1CD-2 subset 1
NKnatural killer

Footnotes
Authorship: Contribution FvR, DEHA, SS, SY, designed the research; FvR, DEHA, SS wrote the paper; SS, SY, XL, JS, MD, AvA, YR, B. Balasa, BG, AGR, and DC performed research, collected data, analyzed and interpreted data; FvR and B. Barlogie enrolled and treated patients on research protocols.
Conflict-of-interest disclosure DEHA, MD, AvA, YR, B. Balasa, BG, AGR, and DC are current or former employees at Facet Biotech Corporation (formerly PDL BioPharma), FvR has received research funding from PDL Biopharma, B. Barlogie has received research funding from Millenium Pharmaceuticals.
1. Child JA, Morgan GJ, Davies FE, et al. High-dose chemotherapy with hematopoietic stem-cell rescue for multiple myeloma. N Engl J Med. 2003;348:1875–83. see comment. [PubMed]
2. Barlogie B, Anaissie E, van Rhee F, et al. Incorporating bortezomib into upfront treatment for multiple myeloma: early results of total therapy 3. Br J Haematol. 2007;138:176–85. [PubMed]
3. Zhan F, Huang Y, Colla S, et al. The molecular classification of multiple myeloma. Blood. 2006;108:2020–8. [PubMed]
4. Moreau P, Voillat L, Benboukher L, et al. Rituximab in CD20 positive multiple myeloma. Leukemia. 2007;21:835–6. [PubMed]
5. Zojer N, Kirchbacher K, Vesely M, Hubl W, Ludwig H. Rituximab treatment provides no clinical benefit in patients with pretreated advanced multiple myeloma. Leuk Lymph. 2006;47:1103–9. [PubMed]
6. Treon SP, Pilarski LM, Belch AR, et al. CD20-directed serotherapy in patients with multiple myeloma: biologic considerations and therapeutic applications. J Immunother. 2002;25:72–81. [PubMed]
7. Almeida J, Orfao A, Ocqueteau M, et al. High-sensitive immunophenotyping and DNA ploidy studies for the investigation of minimal residual disease in multiple myeloma. Br J Haematol. 1999;107:121–31. see comment. [PubMed]
8. Tai YT, Catley LP, Mitsiades CS, et al. Mechanisms by which SGN-40, a humanized anti-CD40 antibody, induces cytotoxicity in human multiple myeloma cells: clinical implications. Cancer Res. 2004;64:2846–52. [PubMed]
9. Tai YT, Li XF, Catley L, et al. Immunomodulatory drug lenalidomide (CC-5013, IMiD3) augments anti-CD40 SGN-40-induced cytotoxicity in human multiple myeloma: clinical implications. Cancer Res. 2005;65:11712–20. [PubMed]
10. Tai YT, Li X, Tong X, et al. Human anti-CD40 antagonist antibody triggers significant antitumor activity against human multiple myeloma. Cancer Res. 2005;65:5898–906. [PubMed]
11. Tassone P, Gozzini A, Goldmacher V, et al. In vitro and in vivo activity of the maytansinoid immunoconjugate huN901-N2′-deacetyl-N2′-(3-mercapto-1-oxopropyl)-maytansine against CD56+ multiple myeloma cells. Cancer Res. 2004;64:4629–36. [PubMed]
12. Stein R, Qu Z, Cardillo TM, et al. Antiproliferative activity of a humanized anti-CD74 monoclonal antibody, hLL1, on B-cell malignancies. Blood. 2004;104:3705–11. [PubMed]
13. Sapra P, Stein R, Pickett J, et al. Anti-CD74 antibody-doxorubicin conjugate, IMMU-110, in a human multiple myeloma xenograft and in monkeys. Clin Cancer Res. 2005;11:5257–64. [PubMed]
14. Ozaki S, Kosaka M, Wakahara Y, et al. Humanized anti-HM1.24 antibody mediates myeloma cell cytotoxicity that is enhanced by cytokine stimulation of effector cells. Blood. 1999;93:3922–30. [PubMed]
15. Hsi ED, Steinle R, Balasa B, et al. Expression of CS1 (SLAMF7) in benign and neoplastic plasma cells: a potential new therapeutic target for the treatment of multiple myeloma. Clin Cancer Res. 2008 In Press.
16. Shaughnessy JD, Jr, Zhan F, Burington BE, et al. A validated gene expression model of high-risk multiple myeloma is defined by deregulated expression of genes mapping to chromosome 1. Blood. 2007;109:2276–84. [PubMed]
17. Tai YT, Tonon G, Leiba M, et al. CS1, a new surface target on multiple myeloma (MM) cells, protects myeloma cells from apoptosis via regulation of ERK1/2, AKT and STAT3 signaling cascades. Blood. 2007;110:40a.
18. Bensinger W, Zonder J, Singhal S, et al. Phase I Trial of HuLuc63 in Multiple Myeloma. Blood. 2007;110:358a.
19. Shi J, Tricot G, Garg T, et al. Bortezomib down-regulates the cell surface expression of HLA class I and enhances natural killer cell mediated lysis of myeloma. Blood. 2008;111:1309–17. [PubMed]
20. Hallett WH, Ames E, Motarjemi M, et al. Sensitization of tumor cells to NK cell-mediated killing by proteasome inhibition. J Immunol. 2008;180:163–70. [PubMed]
21. Drobyski WR, Gottlieb M, Carrigan D, et al. Phase I study of safety and pharmacokinetics of a human anticytomegalovirus monoclonal antibody in allogeneic bone marrow transplant recipients. Transplantation. 1991;51:1190–6. [PubMed]
22. Yaccoby S, Barlogie B, Epstein J. Primary myeloma cells growing in SCID-hu mice: a model for studying the biology and treatment of myeloma and its manifestations. Blood. 1998;92:2908–13. [PubMed]
23. Epstein J, Yaccoby S. The SCID-hu myeloma model. Methods in Molecular Medicine. 2005;113:183–90. [PubMed]
24. Zhan F, Hardin J, Kordsmeier B, et al. Global gene expression profiling of multiple myeloma, monoclonal gammopathy of undetermined significance, and normal bone marrow plasma cells. Blood. 2002;99:1745–57. see comment. [PubMed]
25. Tai YT, Dillon M, Song W, et al. Anti-CS1 humanized monoclonal antibody HuLuc63 inhibits myeloma cell adhesion and induces antibody-dependent cellular cytotoxicity in the bone marrow milieu. Blood. 2008 doi: 10.1182/blood-2007-08-107292. Tai et al. [PubMed] [Cross Ref]
26. Song W, Tai YT, Sasada T, et al. Identification of CS1 Peptides for Induction of Antigen-Specific CTLs in Multiple Myeloma. Blood. 2007;110:481a.
27. Berinstein NL, Grillo-Lopez AJ, White CA, et al. Association of serum Rituximab (IDEC-C2B8) concentration and anti-tumor response in the treatment of recurrent low-grade or follicular non-Hodgkin's lymphoma. Annals of Oncology. 1998;9:995–1001. [PubMed]
28. Shaughnessy J, Zhan F, Huang AY, Barlogie B. In vivo changes in gene expression profiles (GEP) after bortezomib for multiple myeloma: differential effects on plasma cells and micro-environment. J Clin Oncol. 2006;24:7603.
29. Rice A, Dillon M, van Abbema A, Afar D. HuLuc63 in Combination Regimens with Conventional and Targeted Therapies Has Additive and Synergistic Anti-Tumor Activity in Pre-Clinical Models of Myeloma. Blood. 2007;110:742a.
30. Bruno B, Giaccone L, Rotta M, Anderson K, Boccadoro M. Novel targeted drugs for the treatment of multiple myeloma: from bench to bedside. Leukemia. 2005;19:1729–38. [PubMed]